Marco Garofalo
email: marco.garofalo@studenti.unime.itflongo

Zakaria Benomar
email: zakaria.benomar@inria.fr

Francesco Longo

Giovanni Merlino

Nikolaos Georgantas
email: nikolaos.georgantas@inria.fr

Antonio Puliafito
email: apuliafito@unime.it

A Service Bus Architecture for Highly Distributed and Dynamic IoT Environments

Keywords: Smart Cities, Cloud computing, Interoperability

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

I. INTRODUCTION

The proliferation of the Internet of Things (IoT) has revolutionized the way we interact with technology, embedding intelligence into various aspects of our lives. From smart homes and connected vehicles to industrial automation and healthcare systems, IoT has enabled seamless connectivity and automation, promising enhanced efficiency, productivity, and improved quality of life. As the IoT ecosystem continues to expand, the need for efficient and reliable communication mechanisms becomes increasingly crucial.

Traditional centralized communication approaches, which rely on a centralized server to facilitate data exchange between IoT devices, face significant challenges when confronted with the scale and complexity of IoT deployments. Issues such as latency, scalability, network congestion, and vulnerability to single points of failure hinder the optimal functioning of IoT applications. To overcome such challenges, Machineto-Machine (M2M) communications using a service-oriented approach (i.e., SOA) in IoT is a relevant approach. The M2M communication model is a promising paradigm providing a decentralized approach for pairs and systems communication. By leveraging the capabilities of M2M communications and SOA in the IoT field, devices and IoT systems/services can establish direct connections, enabling faster and more efficient data transmission, reducing latency, and enhancing overall system responsiveness, compatibility, and management.

Taking as an example the smart city ecosystem, we are led to deal with a heterogeneous IoT environment. In such a large scale deployment, we may leverage tens, hundreds or even thousands of devices belonging to different broadcast domains while using distinct communication protocols (e.g., HTTP, MQTT, CoAP, etc.). Indeed, we can not think about a smart city deployment on which all the devices belong to the same network/sub-network and use the same protocol. Such constraints lead to the adoption of IoT-based systems in very specific, scale-limited and statically defined scenarios (e.g., Local Area Network (LAN)-based deployments).

To deal with the aforementioned challenges, we propose a system meant to ensure seamless interaction between devices, regardless of their localization, underlying networking configuration (e.g., being behind a NAT), and the used communication protocols. For this purpose, we integrate two systems namely IoTronic (a component of the Stack4Things middleware [START_REF] Merlino | Stack4things: Integrating iot with openstack in a smart city context[END_REF] and the Data eXchange Mediator Synthesizer (DeXMS) [START_REF] Bouloukakis | Automated synthesis of mediators for middleware-layer protocol interoperability in the iot[END_REF]. In particular, IoTronic is used as an IoT Infrastructure Manager (IIM) to deal with all the infrastructurerelated duties (e.g., remote management and programmation). On the other hand, DeXMS takes care of mediating communications between IoT devices/systems that use different protocols.

II. SYSTEMS DESCRIPTION AND INTEGRATION

To enhance the IoT smart environments, we integrated DeXMS and the OpenStack-based system Stack4Things. Fig. 2: An HTTP mediation bus for distributed IoT systems protocols mediation. Fig. 2 conceptually shows an instance of the system bus regrouping a set of geographically distributed devices and systems. The architecture depicted in Fig. 2 defines a common middleware technology in a horizontal layer between IoT devices, systems, and IoT applications. The Cloud-based bus is shown as the horizontal blue cylinder. The vertical green dashed lines represent the OpenStack overlay network that covers both the Cloud and the Edge deployments (i.e., VMs, containers, and remote IoT nodes as reported in [START_REF] Benomar | Cloud-based network virtualization in iot with openstack[END_REF]). We mention that each Cloud tenant may create his/her bus connecting a set of distributed equipment following the as-a-Service (aaS) paradigm. All the devices and systems in the figure (including the DeXMS mediators) are part of the same overlay. The mediation layer which is reported in the figure deals with protocols' adaptation. The overlay links (i.e., green dashed lines) can be thought of as "connections" that are the sources and/or destinations of messages flowing over the bus. Regarding the Edge networking network service, the Neutron ports (i.e., the kernel virtual network TAP interfaces) get instantiated on the data center, precisely, on the machines hosting the WebSockets (WS) tunnel agents (i.e., the blue bashed component in Fig. 1) and then attached to the Neutron virtual switching facilities managed by the Neutron L2 agent (i.e., the Linux bridge in Fig. 1) [START_REF] Benomar | Cloud-based network virtualization in iot with openstack[END_REF]. It is worth mentioning that in this networking design, the switching facilities (i.e., virtual switches) are running on the cloud thus making the system highly scalable for particular hefty configuration requirements. Another advantage of the networking solution is the support of mobile devices/systems. Even if a device that belongs to a user-defined overlay is a mobile one and may experience connectivity changes due to vertical handovers (switching between different connectivity options such as WiFi, 3/4/5G) and thus, an underlay networking configuration change (i.e., IP address modification), the device will remain part of the same virtual network while keeping the same overlay IP address as before the connectivity interruption [START_REF] Benomar | Cloud-based network virtualization in iot with openstack[END_REF]. For protocols' adaptation, DeXMS is the pillar of our mediation layer. In the scenario in Fig. 2, a set of protocols can be translated to the bus protocol (in this case, HTTP). Technically, to instantiate a DeXMS mediator, we use Docker containers. We devote containers' provisioning to the OpenStack Zun subsystem to be compliant with the other services we use (e.g., Neutron) and in particular, the virtual networking solution we described earlier [START_REF] Benomar | Cloud-based network virtualization in iot with openstack[END_REF].

III. TESTBED IMPLEMENTATION

We report in this section the description of a preliminary testbed being used to test the system. The implementation uses an IoT device (a Raspberry Pi) deployed in Paris, France, and managed using S4T. The device is configured to send data periodically (every 30 seconds) to an MQTT broker. To expose the IoT device-generated data through the HTTP bus, a DeXMS mediator is deployed. This mediator is managed through S4T and it is configured to be on a user-defined Neutron overlay network. As a client, we use an HTTP-based service deployed on a Raspberry Pi deployed in Messina, Italy. This later HTTP service (which is part of the same network overlay) can the MQTT data using GET requests in the destination of the DeXMS mediator. The service is configured to get data every 30 seconds, it calculates the average value in an interval of 10 minutes and then adds the value to a graph that can be visualized, in real-time, by users. Fig 3 shows the graph of the data being collected and calculated during 24 hours (23/06/2023). We highlight that the presented solution has been also tested with other protocols (e.g., MQTT to HTTP to CoAP and MQTT to HTTP to SOAP) to be used in a French project called Cloud Platform for Smart City (CP4SC).

IV. CONCLUSION

The management of heterogeneous interconnected things is a novel challenge that has to be addressed to lay down the foundation of a fresh way to manage and live in a (Smart) City. We believe that our system can address a set of challenges in IoT. As a future extension of this work, the reported system will be extensively tested with real-world scenarios related to the CP4SC project. In particular, we will use and adapt this solution to deal with a large-scale Intelligent Transportation System (ITS) as well as a use case related to digital twins.

Fig. 1 Fig. 1 :

 11 Fig. 1: IoTronic, Neutron and DeXMS integration design.

Fig. 3 :

 3 Fig. 3: The HTTP-based visualization service.We devote containers' provisioning to the OpenStack Zun subsystem to be compliant with the other services we use (e.g., Neutron) and in particular, the virtual networking solution we described earlier[START_REF] Benomar | Cloud-based network virtualization in iot with openstack[END_REF].


K Megdouli
email: karima.magdouli@sorbonne-universite.fr

Development of a shock circle model based on the two-dimensional distribution to assess the performance of a supersonic ejector used in refregeration systems

Keywords: Ejector, Velocity distribution, Shock circle I. much

Development of a shock circle model based on the two-dimensional distribution to assess the performance of a supersonic ejector used in refregeration systems

K. Megdouli

INTRODUCTION

In Europe, 40% of the electric energy consumption is due to heating and cooling systems. So, increasing their efficiency is a fateful step to reduce heat losses and carbon dioxide emission [START_REF] Braccio | Mass-flowrate-maximization thermodynamic model and simulation of supersonic real-gas ejectors used in refrigeration systems[END_REF]. The majority of heat pump systems operate with Hydrofluorocarbons (HFCs), such as R134a with Global Warming Potential (GWP) of 1430. To act against global warming and reduce greenhouse gas emissions, the European regulation prohibits the use of certain HFCs according to their GPW. From 2030, the refrigerant authorized on refrigeration installations must have a GWP less than 150. As a result of the legislation, R1234yf is introduced as an environmentally friendly fluid [START_REF] Braccio | Mass-flowrate-maximization thermodynamic model and simulation of supersonic real-gas ejectors used in refrigeration systems[END_REF]. To address the increasing demand for cooling, numerous novel thermodynamic cycles have been devised and analyzed to harness low-level heat sources, including industrial waste heat or solar heat. Among these refrigeration cycles designed for low-grade heat utilization, such as absorption cycles [START_REF] Braccio | Simulation of an ammonia-water absorption cycle using exchanger effectiveness[END_REF], adsorption cycles [3], and ejection cycles [START_REF] Elbel | Review of recent developments in advanced ejector technology[END_REF], ejection cycles have garnered a growing interest. Depending on the specific application, ejector sizes can vary significantly, ranging from several meters for desalinating seawater to just a few centimeters for vehicle air-conditioning systems. In the context of refrigeration, employing ejectors offers several advantages. Firstly, it can minimize the energy losses associated with traditional expansion valves in conventional refrigeration cycles. Secondly, in Ejector Refrigeration Systems (ERS), compression can be achieved using low-grade thermal sources like solar energy or industrial waste heat, thereby reducing reliance on fossil fuels and curbing greenhouse gas emissions.

979-8-3503-4284-0/23/$31.00©2023 European Union

Furthermore, ejectors have the benefit of having no moving parts, making them straightforward to construct, install, and maintain at a low cost. An ejector consists of two nozzles, known as the primary and secondary nozzles, which are designed to converge and then diverge, as depicted in Fig. 1. These two nozzles are aligned, with the primary nozzle situated within the secondary nozzle. The fundamental principle of a supersonic ejector can be summarized as follows [START_REF] Braccio | Simulation of an ammonia-water absorption cycle using exchanger effectiveness[END_REF]: Initially, the high-pressure primary flow (designated as P p and T p ) undergoes expansion as it passes through the primary nozzle, ultimately reaching supersonic speeds at the nozzle's exit. This supersonic flow effectively captures and draws in the secondary flow, which operates at a lower stagnation pressure (P s ). Subsequently, these two flows, characterized by different velocities and temperatures, mix within the mixing duct, resulting in the exchange of both momentum and energy. Finally, the mixed flow undergoes compression within the diffuser section. This entire process serves two primary purposes: firstly, the entrainment of the secondary flow, and secondly, its compression. Consequently, the performance of an ejector is typically evaluated using two distinctive parameters [3]:

-The compression Ratio (r): The compression ratio, denoted as r, represents the relationship between the total pressure at the ejector outlet (P b ) and the pressure at the secondary inlet (P s ). It quantifies the ratio of these two pressure values. -In the on-design region, both the primary and secondary flows are choked, meaning that changes in the back pressure do not impact the entrainment ratio. Importantly, information about the outlet conditions does not affect the upstream choked section. -Beyond a critical point, as the back pressure rises, the entrainment ratio declines because the secondary flow is no longer choked. The ejector operates in the off-design region, and its efficiency decreases as a result. -If the back pressure is further increased past a breakdown pressure, a reverse flow occurs at the secondary inlet, causing the ejector to malfunction. Typically, the choking condition of the secondary flow is identified when the sonic line reaches the ejector wall. At this point, the secondary flow becomes entirely supersonic.

Fig. 2. A graph depicting the performance curve of an ejector

II.

Real gas implementation The selection of the Peng-Robinson Equation of State (PREOS) was motivated by its extensive application within the chemical industry [START_REF] Lopez-Echeverry | Peng-Robinson equation of state: 40 years through cubics[END_REF][START_REF] Julián | Short nozzles design for real gas supersonic flow using the method of Characteristics[END_REF]. Equation ( 1) is utilized to determine the pressure ( ) using coefficients derived from Equation ( 2) for and Equation (3) for . Additionally, the parameters and for a pure substance are computed based on the critical pressure ( ), critical temperature ( ), and acentric factor ( ). MODELING AND ANALYSIS The 2-D flow dynamics equations governing the behavior of an ejector (Fig. 1) are inherently intricate, encompassing principles of mass conservation, momentum conservation, and energy conservation. To facilitate the analysis, certain simplifying assumptions are applied without sacrificing the comprehensiveness of the study. 1-The primary flow inside the ejector is evenly distributed in the radial (r) direction. 3-The pressure and temperature of the secondary flow exhibit uniform distribution along the radial (r) direction. 4-The ratio of the length (L x ) to the diameter (D 2 ) is approximately 1.50, ensuring that the primary flow can expand and fully accelerate the secondary flow within the suction chamber. This causes the secondary flow to reach the choking condition at the cross-section designated as A-A. 5-The pressure of the secondary flow within the suction chamber maintains a uniform distribution and remains equal to its inlet pressure. The mass flow rate of the primary flow through the nozzle (m p ) when it reaches the choking condition can be determined as indicated below [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF]:

1 1 2( 1) 2 2 ( ) ( ) 1 p p p t g p m P A R T (5)
Where, p represents the coefficient that accounts for the isentropic efficiency of compressible flow within the nozzle. P p and T p denote the static pressure and temperature of the primary flow at the nozzle's inlet, respectively. By applying principles of mass conservation, energy conservation, and utilizing isentropic flow equations, we establish the connection between the nozzle's exit diameter (D 1 ), Mach number at the nozzle exit (M 1 ), nozzle throat diameter (D t ), and Mach number at the nozzle throat (M t ) as follows [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF]:

1 1 2 4( 1) 1 1 2 1 2 ( 1) 1 ( ) 2 ( 1) t D M D M (6) 2 1 1 1 ( 1) 2 p t T M T (7) 1 1 1 g V M R T (8)
Considering the temperature (T 1 ) and velocity (V 1 ) of the primary flow at the nozzle exit, and taking into account that the primary flow expands completely within the suction chamber (as per assumption 4), we can represent the ambient pressure of the expanding flow as equivalent to the pressure of the surrounding secondary flow (denoted as P s ). By applying principles of energy conservation and utilizing isentropic flow equations in gas dynamics, we derive the following equations [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF]:

1 2 1 1 ( 1) 2 r p PA s P M P (9) 2 1 1 ( 1) 2 p PA PA T M T (10) PA PA g PA V M R T (11) 1 1 2 4( 1) 1 2 2 1 1 2 ( 1) ( ) 2 ( 1) PA PA PA D M M D M M
(12) Where: the practical effective expanded diameter is linked to the isentropic efficiency ( exp ) within the suction chamber. This relationship is typically expressed as follows, considering the parameters: P 1 (primary flow pressure at nozzle exit), M PA (Mach number), T PA (temperature), V PA (velocity in the x direction) of the primary flow at section A-A, and PA D (effective expanded diameter of the primary flow in an ideal process):

/ PA PA exp D D
(13) Considering that the isentropic coefficient for the primary flow is represented as p and for the secondary flow as s , it's logical to account for the combined impact of friction losses from both the primary and secondary flows within the suction chamber by utilizing a product of p and s .

In other words, this product factorizes the collective effect on friction losses attributed to both flow : exp p s (14) The velocity in the radial (r) direction at section A-A closely resembles an exponential distribution, we can employ the following equation to describe the variation of this variable relationship [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF]:

1 2 (1 ) r n PA v r V R
(15) Where r v is velocity at radius r in the x direction, V pA is velocity of the primary flow in the x direction at section A-A. When we apply the natural logarithm to both sides of the previous equation, we obtain the following result: (20) Using assumption (3) and the preceding equations, we finally obtain [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF]:

1 2 1 2 2 2 2 2 2 2 2 2 2 (1 ) (1 ) 1 2 2 n n PA PA PA n n sA PA V nR R nR R V R R n R n R (21) 1 2 1 2 2 2 2 2 2 2 (1 ) (1 ) 1 2 2 n n s PA PA PA n n s g sA PV nR R nR R m R T n R n R (22)
Ultimately, in the case of an ideal gas, the energy balance relationship can be articulated as follows [START_REF] Aidoun | Current Advances in Ejector Modeling, Experimentation and Applications for Refrigeration and Heat Pumps. Part 1: Single-Phase Ejectors[END_REF]:

2 2 1 1 ( ) ( ) 2 2 p p p s s s p p pA pA s p sA sA loss m c T m c T m c T V m c T V E (23) 2 2 2 1 1 1 1 (1 ) (1 ) (1

IV. RESULTS AND DISCUSSION

A. Validation of the ejector model

The experimental data from Huang et al. [START_REF] Besagni | Ejector refrigeration: A comprehensive review[END_REF] was employed to validate the proposed ejector model, and Table 1 demonstrates a strong concurrence between the two sets of data. By referring to Table 1, we can affirm that the presented model exhibits superior accuracy in its predictions of the ejector's entrainment performance.

B. Response Surface Methodology (RSM) RSM employs a sensible design approach and employs several quadratic regression equations to establish a functional connection between factors and response values. The theoretical examination of crucial structural parameters, especially the intricate effects of multi-parameter interactions on the performance of the ejector within the BER cycle, can be quite complex. Analyzing how each parameter affects the ejector's performance allows us to discern the trend of their respective influences on the ejector's performance. [START_REF] Huang | A 1-D analysis of ejector performance[END_REF] In this study, the nozzle throat diameter D t ranged from 1.86 mm to 3.64 mm, while the exit nozzle diameter D 1 varied between 3.5 mm and 6.5 mm. The boundary conditions for the ejector remained constant throughout our simulations, including a primary flow inlet temperature of 90°C, a primary flow inlet pressure of 5.38 bar, a secondary flow inlet pressure of 0.434 bar, a secondary flow inlet temperature of 10°C, and an ejector outlet pressure of 1.12 bar. The quadratic representation of this correlation can be found in eqn. 25:

2 0 1 1 n n i i ii i ij i j i i ij y x x xx ( 25
)
In this equation, y represents the output variable (U), β signifies the coefficient, x stands for the input variable (namely, D t and D 1 ), and ε denotes the error in observations. Fig. 2 shows the changes in the RSM for ejector performance at various D t , and D 1 . We found that the ejector's performance is notably responsive to variations in D t , as evidenced by the steepness of its contour line. However, it was found that there exists an optimal value for D t , which led to the optimization of the ejector. To delve deeper into the impact of this parameter on the ejector's performance, individual variable analyses is conducted.

During the single-variable analysis, it is necessary to maintain the other variable constant, D 1 = 4.5 mm. The RSM analysis of the data indicates a strong quadratic relationship with a favorable regression coefficient between the independent factor variables and U. The quadratic polynomial equation for U, which incorporates the independent variables, can be formulated as follows:

C. Sensitivity analysis

After validating the ejector model, it is implemented in an ERS driven by low-grade heat sources to enhance its performance, Fig. 5. This integration enables the refrigeration system to operate more sustainably and economically, making it a viable and efficient solution for cooling applications powered by low-grade heat [START_REF] Megdouli | Optimization of carbon dioxide ejector expansion transcritical refrigeration system with ANOVA and NSGA-II[END_REF]. The successful application of the ejector model contributes to the overall advancement and innovation of environmentally friendly and energy-efficient refrigeration technologies.

The coefficient of performance (COP) of the ERS studied is given by the following expression: V. CONCLUSIONS The impact of geometry on ejector performance has been assessed in order to optimize the design of the ejector. Through the application of Response Surface Methodology for investigating the impact of two parameters (D t and D 1 ) on ejector performance, we determined that optimizing the value of the diameter of the nozzle throat could enhance ejector performance. The sensitivity analysis demonstrated that the sensitivities of ejector performance to these parameters followed a descending order: D t >D 1 . Consequently, when aiming to optimize ejector performance, D t should take precedence. These results provide clarity regarding the hierarchy and influence of multiple variables in the analyses. Following the validation of the ejector model, it is integrated into a refrigeration system powered by low-grade heat sources to improve its operational efficiency. The influence of design parameters on the thermodynamic performance of the system studied is investigated. It is observed that when the system operates with a generator temperature of 90°C, an evaporation temperature of 12 and a condensation temperature equal to 35°C, the system has an efficiency of 0.31. We can confirm that the ejector refrigeration system demonstrates a high level of competitiveness and efficiency.

-

  The entrainment Ratio (U): The entrainment ratio, symbolized as U, signifies the ratio between the mass flow rate of the secondary flow (2

Fig. 1 .

 1 Fig. 1. Schematic diagram of the ejector Fig. 2 displays a graph illustrating the behavior of an ejector, specifically showing the entrainment ratio in relation to the back pressure. This curve reveals three distinct operational zones when maintaining constant inlet pressures (Both primary and secondary flow) and gradually increasing the back pressure.-In the on-design region, both the primary and secondary flows are choked, meaning that changes in the back pressure do not impact the entrainment ratio. Importantly, information about the outlet conditions does not affect the upstream choked section. -Beyond a critical point, as the back pressure rises, the entrainment ratio declines because the secondary flow is no longer choked. The ejector operates in the off-design region, and its efficiency decreases as a result. -If the back pressure is further increased past a breakdown pressure, a reverse flow occurs at the secondary inlet, causing the ejector to malfunction. Typically, the choking condition of the secondary flow is identified when the sonic line reaches the ejector wall. At this point, the secondary flow becomes entirely supersonic.

  To determine the value of n, we should utilize the conditions present at the shock circle. The velocity of sound at the shock circle is[START_REF] Lopez-Echeverry | Peng-Robinson equation of state: 40 years through cubics[END_REF]: Define the mean flow velocity and the mass flow rate of the secondary flow at section A-A as follows[START_REF] Lopez-Echeverry | Peng-Robinson equation of state: 40 years through cubics[END_REF]

Fig. 3 .

 3 Fig. 3. Response surface analyses of the entrainment ratio with Dt and D1

Fig. 4 .

 4 Fig. 4. Variation in U with Dt

Fig. 5 .

 5 Fig. 5. Schematic of an ejector refrigeration system (ERS) Fig. 6 illustrates the impact of the evaporator temperature

Fig. 6 .

 6 Fig. 6. Variation in the COP with the evaporator temperature for different generator temperatures at Tcond = 35 °C

TABLE 1 :

 1 THE OUTCOMES OF OUR CURRENT STUDY ARE JUXTAPOSED WITH THE RESULTS DERIVED FROM THE EXPERIMENTAL DATA PRESENTED BY HUANG ET AL.

  Fig.4depicts how the ejector's performance changes with varying values of D t (nozzle throat diameter). Initially, as D t increases, the ejector performance improves, but after reaching a certain point, it starts to decline significantly. Subsequently, U experiences a rapid increase as D t continues to rise. The maximum U value of 0.353 is achieved when D t is set at 2.66 mm, and afterward, it begins to decrease with further increases in D t . This illustrates that within the range of nozzle throat diameter variations, there is a substantial U variation of approximately 116%, highlighting the significant influence of D t on the ejector's performance.

						(25)
	U	0.3538 0.0006	t D	1 D 0.0001	0.0069	2 t D

Authorized licensed use limited to: Princess Sumaya University for Technology. Downloaded on January 19,2024 at 17:32:19 UTC from IEEE Xplore. Restrictions apply.

ACKNOWLEDGMENTS

The author thanks the Institut Jean Le Rond D'Alembert at Sorbonne Université for its support with the dissemination of this research work.


Marianne Bernard
email: marianne.bernard@univ-poitiers.fr

Pierre Voisin

Photoreceptor-specific expression, light-dependent localization and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina

Keywords: Yin Yang 1, chicken, retina, photoreceptor, hydroxyindole-O-methyltransferase, melatonin

Photoreceptor-specific expression, light-dependent localization, and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina

INTRODUCTION

Yin Yang 1 (YY1) is an ubiquitous zinc-finger protein of the Polycomb group (PcG) that can act either as an activator or as a repressor of gene transcription, depending on promoter and cellular context (reviewed in: [START_REF] Shi | Everything you have ever wanted to know about Yin Yang 1[END_REF][START_REF] Thomas | Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key?[END_REF][START_REF] Akopyan | Transcription factor YY1: structure, function, and therapeutic implications in cancer biology[END_REF].

YY1 is multifunctional and plays critical roles in different aspects of cell biology, including proliferation, differentiation and apoptosis [START_REF] Akopyan | Transcription factor YY1: structure, function, and therapeutic implications in cancer biology[END_REF]. As a regulator of cell proliferation, YY1 has been shown to act either through transcriptional activation of cellcycle progression genes, such as c-myc [START_REF] Riggs | Yin-yang 1 activates the c-myc promoter[END_REF], or through direct interaction with-and resulting inactivation of-the tumor suppressor gene product p53 [START_REF] Sui | Yin Yang 1 is a negative regulator of p53[END_REF].

The actions of YY1 on key regulators of cell proliferation and apoptosis have led to the notion that it may act as an initiator of tumorigenesis, and may therefore constitute an effective target for anti-cancer therapy [START_REF] Akopyan | Transcription factor YY1: structure, function, and therapeutic implications in cancer biology[END_REF]. Beside its implication in tumorigenic proliferation, YY1 plays a fundamental role in physiological processes that are crucial for normal embryonic development. As a clear indication of this, targeted disruption of the YY1 gene in mice resulted in peri-implantation lethality [START_REF] Donohoe | Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality[END_REF]. Consistent with a role of YY1 in the control of cell division, analysis of heterozygote (YY1+/-) mice revealed that a subset of these animals showed dramatic growth retardation. In addition, these mice displayed brain defects that suggested YY1 was required for normal progress of neurulation [START_REF] Donohoe | Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality[END_REF]. This was further supported by developmental studies in Xenopus, where overexpression of YY1 in one blastomere of two-cell stage embryos, induced the formation of an ectopic neural axis [START_REF] Satijn | The polycomb group protein EED interacts with YY1, and both proteins induce neural tissue in Xenopus embryos[END_REF]. During Xenopus development, YY1 was found to be predominantly expressed in the anterior neural tube, developing brain and optic vesicles [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF]. Inhibition of YY1 synthesis by injection of antisense oligonucleotides in Xenopus embryos caused a slight developmental delay and a marked reduction in the size of head structures and eyes [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF]. Additional evidence involving YY1 in eye development came from a study showing that the abnormal anterior eye formation observed in Ring1-deficient mice was exacerbated in compound YY1+/-Ring1-/-mice [START_REF] Lorente | Homeotic transformations of the axial skeleton of YY1 mutant mice and genetic interaction with the Polycomb group gene Ring1/Ring1A[END_REF]. A similar phenotype was recently reported in Rybp (Ring1 and YY1 binding protein) heterozygous null mouse embryos [START_REF] Pirity | Rybp, a polycomb complex-associated protein, is required for mouse eye development[END_REF]. A result suggesting that Rybp, Ring1 and YY1 may interact to orchestrate normal vertebrate eye development. In addition, in Xenopus and mouse embryos YY1 has been shown to activate the expression of Otx2 [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF][START_REF] Takasaki | Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart[END_REF], a homeobox transcription factor that plays a central role in retinal cell differentiation [START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland[END_REF][START_REF] Viczian | XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina[END_REF]. The accumulating evidence relating YY1 to eye development prompted us to explore the hypothesis that YY1 may play a role in regulating retina-specific gene expression. In the present work, we examined the expression of YY1 in the chicken retina and we observed that this transcription factor is mainly expressed in photoreceptors. Functionally, we show that YY1 binds to regulatory regions of several genes specifically expressed in photoreceptors and we demonstrate that the YY1-binding site contained in the promoter of the gene encoding the last enzyme of the melatonin-synthesis pathway, hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4), is required for full transcriptional activation in chicken embryonic retinal cells.

MATERIALS AND METHODS

Animals

Fertilized chicken eggs (Gallus domesticus) were purchased from Rumulo C ie (Quinçay, France) and were incubated at 37°C in an atmosphere of 70 % humidity. Embryos were staged according to [START_REF] Hamburger | Series of embryonic chicken growth[END_REF].

Adult chickens (Gallus domesticus) were purchased at 8 weeks of age from Lecorne C ie (Lencloître, France) and were kept for one week in a 12h light/12h darkness photoperiod (lights on zeitgeber time (ZT) 0-12), in a room at 27°C, with food and water ad libitum. They were anesthesized by an intraperitoneal injection of chloral hydrate and were sacrificed by decapitation, either at ZT23 (dark-adapted chickens, n = 3) or at ZT11 (light-adapted chickens, n = 3). Following dissection, tissues were either transferred in culture medium (for retinal cell culture) or frozen in liquid nitrogen (for western blot analysis) or fixed in Bouin's solution (for immunohistochemistry). Animal care was conducted in accordance with the standard ethical guidelines (European Community Guidelines on the Care and Use of Laboratory Animals; and French Agriculture and Forestry Ministry guidelines for handling animals, decree 87848, license E 86-040) and approved by the local ethical committee.

Immunohistochemistry (IHC)

Retinas fixed in Bouin's solution were dehydrated in ethanol, then butanol, and embedded in paraffin. Sections (6 µm) were blocked in phosphate-buffered saline containing 0.05% Tween-20 (PBS-Tween), 1% BSA, 1% gelatin and were incubated overnight at 4°C with a rabbit polyclonal anti-YY1 antibody (Santa-Cruz, sc-281) diluted 1:1000 in PBS-Tween, 1% BSA, or with a rabbit polyclonal anti-LexA antibody (Affinity Bioreagents, PA1-4966) at the same dilution. The primary complexes were detected by incubation with biotinylated goat anti-rabbit IgG (Vector laboratories) diluted 1/500 in PBS-Tween and with avidin-biotinperoxidase complexes ("Vectastain" Kit, Vector Laboratories). Peroxidase activity was revealed with 0.05% diaminobenzidine, 38mM ammonium nickel sulfate and 0.003% H2O2 in 0.15M sodium acetate and 3.7mM acetic acid. Sections were observed on a light microscope (Zeiss Axioplan) equipped with a digital camera (Kappa).

Western blots

Protein extracts were prepared by sonicating the frozen tissues in phosphate buffer. After quantitation with Bradford dye (Bio-Rad), proteins were diluted in Laemmli buffer, fractionated by SDS-PAGE (8% gels) [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]) and electrotransferred on .nitrocellulose membrane. Non-specific sites were blocked in PBS-Tween with 1% BSA and 4% ProtoBlock TM (National Diagnostics) and the membranes were incubated overnight at 4°C with the anti-YY1 or anti-LexA antibodies used for IHC, or with a mouse monoclonal antibeta-tubulin antibody (Sigma, T4026) diluted 1:400 in PBS-Tween, 1% BSA. For YY1 and LexA, antibody-antigen complexes were detected as described for IHC. For beta-tubulin, antibody-antigen complexes were detected with peroxidase-linked sheep F(ab')2 fragments anti-mouse IgG (Amersham Biosciences) and peroxidase activity was detected as described for IHC.

Cloning of the full-length chicken YY1

The full-length chicken YY1 cDNA (GenBank accession number AJ719349) was obtained by RT-PCR from total RNA of chicken retina, with a sense primer containing the Kozak sequence and the first 14 bases of the YY1 coding sequence, with a KpnI site added at its 5'end ( 5' -CCTGGTACCGCCATGGCCTCGGGAGA-3' ) and an antisense primer containing the last 21 bases of the YY1 coding sequence, with a BamHI site added at its 5'-end ( 5' -TCCGGATCCTTTCACTGGTTGTTTTTGGCCTT-3' ). The PCR product obtained was cloned into pcDNA3 (InVitrogen) and sequenced with Big Dye reagent (ABI) (analysis on ABI Prism 310).

Electrophoretic mobility shift assay (EMSA)

Based on the consensus YY1-binding sequence published on the TESS software (Transcription Element Search Software on the www ; TRANSFAC matrix record M00059), eight putative YY1-binding sites were identified in different regulatory regions of genes specifically expressed in photoreceptors (Table 1). These sequences were analyzed by electrophoretic mobility shift assay (EMSA) as previously described [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF][START_REF] Dinet | Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2[END_REF]. Proteins were extracted from chicken retinas, rat retinas and Y79 human retinoblastoma cells (ECACC). Recombinant chicken YY1 protein was synthesized in vitro with the transcription/translation-coupled reticulocyte lysate system (TNT  , Promega), using the pcDNA3-YY1 plasmid described above as template. The double-stranded oligonucleotides used as DNA probes corresponded to the sequences described in Table 1 and were labelled with [-32 P]ATP (3000 Ci/mmole) using T4 polynucleotide kinase (Promega).

The DNA-protein binding assay was carried out for 15 min at room temperature, with 5 fmoles of [ 32 P]-labelled DNA probe (30, 000 cpm) and with either in vitro-translated YY1 (4 µl of a TNT  reaction) or 20 µg of protein extracts, in the absence or presence of a molar excess of non-radioactive DNA competitor (see figure legends for details). The final composition of the reaction was as previously described [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF]. In some reactions, the proteins were preincubated with 2 µl of anti-YY1 antibody or 2 µl of normal rabbit IgG for 45 min at room temperature before adding the radiolabelled probe. The DNAprotein complexes were analyzed on non-denaturing gels [4.5% acrylamide/bisacrylamide (30 : 1) in 40 mM Tris, 270 mM glycine and 2 mM EDTA]. The gels were dried and exposed overnight to a PhosphorImager screen (Storm 820, Molecular Dynamics). The images were analyzed with the ImageQuant program (Molecular Dynamics).

Cell culture and transient transfections

HEK293 cells (a generous gift from the Laboratory of Prof. J. M. Müller, Poitiers) were maintained in Dulbecco's modified Eagle's medium supplemented with 100 U/mL penicillin/streptomycin and 10% fetal calf serum (Gibco BRL). Primary cultures of chicken embryonic retinal cells were prepared as previously described [START_REF] Dinet | Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2[END_REF], with slight modifications. Embryonic day 8 (E8) neural retinas, free of pigment epithelial cells, were dissociated and cultured in polyornithine-coated plates, in M199 medium (Sigma) supplemented with 100 U/mL penicillin/streptomycin, 2 mM glutamine, 0.1% bovine serum albumin, and 10% newborn calf serum (Gibco BRL). HEK293 and retinal cells were incubated at 37°C in an atmosphere of 5% CO2 in air. They were seeded in 6-well plates, one day before transfection, at a density of 2 x 10 5 cells per well for HEK293 cells, or 3 x 10 6 cells per well for retinal cells. Cells were transfected by the CaPO4 method. Briefly, appropriate amounts of plasmid DNA (see Figure legends) were diluted in 75 µl of 0.25M CaCl2 and were added dropwise to 75 µl of 2xHeBS (280 mM NaCl, 1.4 mM Na2HPO4, 50 mM HEPES, pH 7.1). Cells were incubated overnight with the precipitate (150 µl/well) and were cultured for 24 h in fresh medium before being assayed for luciferase activities. Transfections and assays were performed in triplicate for each experimental group.

Three categories of plasmids were used for transient transfection experiments. HEK293 cells and retinal cells were transfected with the pGL3-HIOMT promoter-reporter construct, containing 3 kb of the chicken HIOMT promoter upstream of the firefly luciferase reporter gene (described as pGL3A in [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF]. The pGL3-HIOMT(cH1mut) corresponded to pGL3-HIOMT with a mutation in the YY1-binding site (CCCATTT → mutated plasmids were controlled by direct sequencing. The pcDNA3-YY1 plasmid described above was used to overexpress YY1 in transfected HEK293 cells. A plasmid expressing the rat Crx transcription factor [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF] was used as a positive control and the empty pcDNA3 vector was used as a negative control. Corrections for variations in transfection efficiencies were performed with pRL-SV40 (Promega), a plasmid expressing the Renilla luciferase under control of the SV40 promoter end enhancer.

Firefly and Renilla luciferase activities, were assayed using the Dual-Glo TM luciferase assay system (Promega). Light emission was measured on a Beckman LS 6500 counter, on the luminometer program (Beckman Instruments). Transfection efficiency was normalized to the firefly/Renilla luciferase activity ratio. The statistical significance of the results were verified by the Student's t-test.

RESULTS

Tissue specificity of YY1 expression

Western blot analysis of a series of chicken tissues indicated that YY1 is expressed predominantly in the neural retina, retinal pigment epithelium (RPE), pineal gland and duodenum (Fig. 1A). Lower expression levels could be detected in telencephalon, cerebellum, liver and heart, while YY1 remained undetectable in skeletal muscle (Fig. 1A). In agreement with previous reports in human and rat [START_REF] Shi | Everything you have ever wanted to know about Yin Yang 1[END_REF][START_REF] Ellis | Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1[END_REF], YY1 was detected as a 63 kDa protein on western blots. Depending on tissues, some amount of non-specific reaction could be observed, as determined with an irrelevant antibody (Fig. 1A). The neural retina stood out as the tissue where 63 kDa YY1 was detected with the highest specificity (Fig. 1A). As illustrated in figure 1B, the YY1 protein was readily detectable in the retina of 6day embryos (E6). The signal gradually decreased until day 1 post-hatch (P1), then stabilized between P1 and P15 (Fig. 1B). The use of tubulin as an internal standard confirmed the decrease in YY1 intensity was real and did not reflect differences in the total amount of protein loaded on the gel (compare E6 and P15 in Fig. 1B).

Cellular localization of YY1 in dark-adapted retina

Immunohistochemical detection of YY1 in the retina of dark-adapted chickens was highly specific of photoreceptors (Fig. 2A,B). In the central retina, YY1 labelling was mainly concentrated in inner segments above the outer limiting membrane (Fig. 2A,C), while in more peripheral zones it gradually extended to the outer nuclear layer and to the basal pedicles of photoreceptors (Fig. 2B, D-I). Different morphological subtypes of photoreceptors were observed. The strongest labelling was found in accessory cones of the abundant doublecones, particularly in the myoid region and around the large paraboloid and ellipsoid regions (Fig. 2C-H). Some accessory cones displayed strong reactions in the basal pedicle and nucleus (Fig. 2D,E,F,H) and some were also intensely labelled at the apex of the inner segment, just proximal to the lipid droplet (Fig. 2F-H). Principal cones of double-cones showed a more diffuse and weaker labelling than accessory cones, but some had a distinct nuclear labelling (Fig. 2C,G). YY1 labelling could also be identified in rod nuclei, typically localized at the vitreal side of the outer nuclear layer (Fig. 2I,J). The number of labelled photoreceptor nuclei and the intensity of the nuclear labelling showed large individual and zonal variations. YY1 was not detected in outer segments, but was found in the calycal process of some photoreceptors (Fig. 2C,E,I).

Upon extended development of the chromogen, a low intensity labelling could be observed in some nuclei of the inner nuclear layer and in the cytoplasmic extensions of Müller glial cells (Fig. 2K). This low intensity labelling was not background noise, because it was not observed with an irrelevant antibody (Fig. 2L).

Effect of light on the subcellular localization of YY1

When YY1 immunohistochemical labelling was compared in light-adapted and dark-adapted retinas, striking differences could be observed in the subcellular localization of the protein (Fig. 3, compare A and B ; also compare to dark-adapted retina in Fig. 2). Light-adapted retinas displayed a spatially more restricted YY1 labelling, mostly overlying the outer limiting membrane (Fig. 3A). In accessory cones of double-cones, YY1 was still present in the myoid region of the inner segment, but the intense labelling around the paraboloid/ellipsoid region and in the basal pedicle was no longer observed (Fig. 3C).

Principle cones and single cones displayed a more diffuse labelling in the inner segment.

Labelled photoreceptor nuclei were still observed in light-adapted retina (Fig. 3C). Our attempts to quantify light-induced differences in the number of YY1-immunoreactive photoreceptor nuclei remained inconclusive, due to large individual variation. As in darkadapted retinas, a low-level labelling could be observed in the inner nuclear layer of lightadapted retinas upon extended chromogen development (data not shown).

Western blot analysis of dark-adapted versus light-adapted retinas revealed no significant differences in the amount or size of the YY1 protein (Fig. 3D).

Interaction of YY1 with regulatory regions of genes expressed in photoreceptors

Strong YY1 immunochemical labelling in the nuclei of some rods and cones prompted us to examine YY1 binding to promoter regions of genes specifically expressed in photoreceptors.

Based on the consensus sequence for YY1 binding elements (see Materials and Methods for details), putative binding sites could be identified in the chicken rhodopsin promoter, as well as in the promoters of two genes of the melatonin pathway, arylalkylamine Nacetyltransferase (AANAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT) (Table 1). Using EMSA analysis, only one element of the chicken HIOMT promoter (cH1) showed saturable, sequence-specific interaction with a protein from chicken retina (Fig. 4A andB). In contrast, the other putative sites found in HIOMT, AANAT and rhodopsin promoters gave non-specific shifted bands of weak intensity, that were not displaced by the unlabelled homologous probes (Fig. 4A). YY1 binding to the cH1 element was further characterized. The retinal protein that bound to cH1 could be identified as YY1, because its binding to DNA was specifically inhibited by an anti-YY1 antiserum, raised against the Cterminal zinc-finger domain of YY1 (Fig. 4C). Moreover, the cH1 element clearly bound the full-length in vitro-expressed chicken YY1 protein (Fig. 4C).

Four additional consensus sites were found in photoreceptor-expressed genes of other species (Table 1). EMSA analysis with anti-YY1 antibody interference positively identified two of these sequences as YY1-binding sites: one in the first intron of the rat AANAT gene, the other one in the 5'-flanking region of the human cone alpha-transducin gene (Fig. 4D). A result suggesting that YY1 may target photoreceptor-expressed genes in different species.

To keep our focus on the chicken retina, where photoreceptor-specificity and light-dark regulation of YY1 were clearly demonstrated, we moved on to examine the functional impact of YY1 on the chicken HIOMT promoter. HEK293 cells were cotransfected with a promoterreporter plasmid containing a 3kb version of the chicken HIOMT promoter and with an expression plasmid encoding chicken YY1. Although transfection of HEK293 cells led to overexpression of the YY1 protein, as verified by western blot (data not shown), the HIOMT promoter remained inactive in these cells (Fig. 4E, left panel). In contrast, we verified that the rat Crx transcription factor was able to activate the HIOMT promoter in HEK293 cells (Fig. 4E, left panel), as previously shown in COS7 cells [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF]. Although YY1 was apparently not sufficient to directly activate the HIOMT promoter, a role of the YY1-binding site contained in this promoter could be clearly established upon transfection of the promoterreporter plasmid into chicken embryonic retinal cells. Indeed, a single-nucleotide mutation that abolishes YY1-binding in EMSA (Fig. 4B), also reduced by 70% the HIOMT promoter activity (Fig. 4E, right panel).

DISCUSSION

The present study provides the first evidence of a photoreceptor-specific expression of YY1 in the chicken retina and further shows that light causes a subcellular translocation of YY1 in photoreceptors. In addition, we demonstrate that YY1 binds to regulatory regions of genes specifically expressed in photoreceptors and we show that its binding-site in the promoter of one of these genes -HIOMT-is required for full transcriptional activity in embryonic chicken photoreceptors.

The first analyses of YY1 expression by whole-mount in situ hybridization in mouse embryos have led to the description of YY1 as an ubiquitous protein [START_REF] Donohoe | Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality[END_REF]. This notion was somewhat revised in Xenopus embryos, where YY1 expression was found to be ubiquitous in early embryos, but progressively restricted to anterior neural structures during later embryonic stages [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF]. In keeping with these results, our western blot analysis indicates a relatively widespread tissue distribution of YY1 in adult chicken, with neural retina beeing one of the tissues with highest YY1 expression level. More surprisingly, our immunohistochemical examination of the retinal neuroepithelium revealed that YY1 is not present in all chicken retinal cells, but is predominantly expressed in photoreceptors. Although an implication of YY1 in normal eye development has been suggested in different studies [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF][START_REF] Lorente | Homeotic transformations of the axial skeleton of YY1 mutant mice and genetic interaction with the Polycomb group gene Ring1/Ring1A[END_REF][START_REF] Pirity | Rybp, a polycomb complex-associated protein, is required for mouse eye development[END_REF], the cellular and molecular basis of this action still remain unknown. The data presented herein suggest that this action of YY1 may include a specific role on photoreceptor differentiation and/or homeostasis. The contrast observed here between ubiquitous tissuedistribution and high cellular-specificity in the retina warrants further immunohistochemical studies in different tissues, aimed at identifying the YY1-expressing cell types. Because partial inactivation of YY1 causes strong defects in neurulation and/or eye development in mouse and Xenopus [START_REF] Donohoe | Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality[END_REF][START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF], the immunohistochemical identification of YY1-expressing cells in the brain and eyes of these animals would be worthwhile, as it should help specify the mechanisms involved in these defects.

Another intriguing aspect of our findings is the effect of light on the subcellular localization of YY1 in photoreceptors. Consistent with previous observations in other cell types [START_REF] Ficzycz | Expression, activity, and subcellular localization of the Yin Yang 1 transcription factor in Xenopus oocytes and embryos[END_REF][START_REF] Palko | The Yin Yang-1 (YY1) protein undergoes a DNA-replication-associated switch in localization from the cytoplasm to the nucleus at the onset of S phase[END_REF] we found that YY1 was not exclusively nuclear. In most photoreceptors indeed, the protein was very abundant in the inner segment and in the basal pedicle. However, we observed that the spatial distribution of the protein was strikingly narrowed following light-adaptation. These changes in YY1 localization are apparently not related to degradation of the protein or to major post-translational modifications, because the size of the immunoreactive YY1 is the same in dark-adapted and light-adapted retinas. The light-induced redistribution of the protein is most visible in accessory cones of double-cones, and is mainly characterized by a concentration of the protein within the myoid region of the voluminous inner segment. This cellular compartment has previously been shown to contain actin filaments that are involved in the day/night photomechanical movements of rods and cones [START_REF] O'connor | Elevation of cyclic AMP activates an actin-dependent contraction in teleost retinal rods[END_REF]. In addition, the myoid cytoskeletal proteins contribute to the subcellular trafficking of proteins in photoreceptors. In mouse, rat, chicken and Xenopus, several phototransduction proteins have been shown to shuttle between the inner and outer segments, on a day-night basis. Transducin and MEKA are translocated from the inner to the outer segment in response to darkness, while arrestin is translocated to the outer segment in response to light [START_REF] Brann | Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina[END_REF][START_REF] Kuo | Translocation of a photoreceptor-specific MEKA protein by light[END_REF][START_REF] Mcginnis | Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells[END_REF][START_REF] Zhu | Mouse cone arrestin expression pattern: light induced translocation in cone photoreceptors[END_REF]. In Xenopus retina, these movements were clearly shown to be controlled by the cytoskeletal network, because they were affected by pharmacological agents that disrupt microfilaments and microtubules [START_REF] Peterson | A role for cytoskeletal elements in the light-driven translocation of proteins in rod photoreceptors[END_REF]. Together, these data suggest that the subcellular trafficking of YY1 may rely on a similar interaction with myoid cytoskeletal proteins. Further studies utilizing cytoskeleton depolymerizing agents will be needed to elucidate this point and to determine the possible consequences of YY1 trafficking in the cytoplasm of photoreceptors. Previous studies in smooth muscle cells have indicated that an interaction of YY1 with polymerized -smooth muscle actin could sequester YY1 in the cytoplasm and thereby inhibit its nuclear translocation [START_REF] Favot | Cytoplasmic YY1 is associated with increased muscle-specific gene expression[END_REF]. We did not find evidence of a similar regulation in our study, because the number of YY1-containing nuclei was not significantly different between light-adapted and dark-adapted photoreceptors.

However, this lack of significance should be re-evaluated in dedicated studies utilizing a larger number of animals and a more quantitative method for immunodetection.

The high level of YY1 expression in the chicken retina and its marked photoreceptorspecificity raise one major question: what role might this transcription factor play in retinal physiology? Considering its well-established role on cell cycle control, the first hypothesis that comes to the mind is that of a contribution of YY1 to retinoblast proliferation. The studies performed in Xenopus embryos favoured this assumption, because they showed that inactivation of YY1 in the embryo generated microphthalmia [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF].

Based on this result, we may surmise that YY1 could play a similar role in the chicken retina.

However, here we detected YY1 in the nuclei of mature photoreceptors, a result indicating that the role of YY1 may not be restricted to the maintenance of retinal precursors in a proliferative status. Rather, the presence of YY1 in the nuclei of fully differentiated photoreceptors would suggest a role of this transcription factor in photoreceptor-specific gene expression. The data presented here support this hypothesis, by showing that YY1 can bind to the regulatory regions of genes specifically expressed in photoreceptors. Moreover, we demonstrate that the promoter of one of these genes (HIOMT) requires an intact YY1 binding-site in order to be fully activated in embryonic photoreceptors. Since HIOMT is the enzyme that catalyzes the last step of synthesis, we suggest that one contribution of YY1 to retinal function might be to favour the production of melatonin by photoreceptors.

However, it seems likely that YY1 would act in conjunction with other transcription factors, because we found that YY1 alone was not to activate the HIOMT promoter in HEK293 cells. Homeodomain-containing factors of the Crx/Otx family might be involved in this process, because they are known to play a major role in the differentiation and maintenance of the photoreceptor phenotype [START_REF] Furukawa | Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation[END_REF][START_REF] Furukawa | Retinopathy and attenuated circadian entrainment in Crx-deficient mice[END_REF][START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland[END_REF][START_REF] Viczian | XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina[END_REF], and we showed in previous studies that they could directly activate the chicken HIOMT promoter [START_REF] Bernard | Transcriptional regulation of the chicken hydroxyindole-O-methyltransferase gene by the cone-rod homeobox-containing protein[END_REF][START_REF] Dinet | Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2[END_REF]. Of course, additional factors expressed in chicken photoreceptors could also be implicated, for instance Optx2 [START_REF] Toy | The Optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes[END_REF], NeuroD [START_REF] Yan | NeuroD induces photoreceptor cell overproduction in vivo and de novo generation in vitro[END_REF][START_REF] Yan | Requirement of neuroD for photoreceptor formation in the chick retina[END_REF], Ngn2 [START_REF] Yan | Neurogenin2 elicits the genesis of retinal neurons from cultures of nonneural cells[END_REF] and RaxL [START_REF] Ohuchi | Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development[END_REF][START_REF] Chen | The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation[END_REF]. Even though HIOMT is the only gene identified here as a potential YY1 target in the chicken retina, we cannot exclude an involvement of YY1 in the transcription of other genes that define photoreceptor-specific functions, particularly those encoding phototransduction proteins. The possibility of an indirect influence of YY1 on photoreceptor-specific transcription should also be hypothesized, because YY1 has been shown to be an essential activator of Otx2 expression in anterior neuroectoderm [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF][START_REF] Takasaki | Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart[END_REF], and Otx2 has the ability to directly activate the promoter of genes specifically expressed in photoreceptors: interphotoreceptor retinoid-binding protein [START_REF] Bobola | OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression[END_REF] ; Crx [START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland[END_REF] ; rhodopsin and M-cone opsin [START_REF] Peng | Chromatin immunoprecipitation identifies photoreceptor transcription factor targets in mouse models of retinal degeneration: New findings and challenges[END_REF], HIOMT [START_REF] Dinet | Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2[END_REF] and rhodopsin kinase [START_REF] Young | Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene[END_REF].

In the light of recent data suggesting an involvement of YY1 in eye development [START_REF] Kwon | Yin Yang 1, a vertebrate polycomb group gene, regulates antero-posterior neural patterning[END_REF][START_REF] Lorente | Homeotic transformations of the axial skeleton of YY1 mutant mice and genetic interaction with the Polycomb group gene Ring1/Ring1A[END_REF][START_REF] Pirity | Rybp, a polycomb complex-associated protein, is required for mouse eye development[END_REF] and based on the results of our study demonstrating the strong YY1 expression in retinal photoreceptors, it might now be interesting to re-examine the retinas of YY1+/-heterozygote mice and of YY1-deficient Xenopus eyes. This would help determine whether decreased YY1 levels in the retina affect photoreceptor-specific gene expression and, as a consequence, photoreceptor differentiation.

Table 1. Sequences of putative YY1-binding elements in photoreceptor-expressed genes.

Based on the consensus YY1-binding sequence defined in the TESS software, eight putative YY1-regulatory elements were identified in the 5'-upstream regions and intronic regions of genes specifically expressed in photoreceptors. The GenBank accession numbers of these regulatory regions are: AF116454 (chicken HIOMT promoter) ; AF193072 (chicken AANAT promoter) ; D00702 (chicken rhodopsin promoter) ; U77455 (rat AANAT promoter and intron 1) ; contig AC091784 and [START_REF] Zhu | Mouse cone arrestin expression pattern: light induced translocation in cone photoreceptors[END_REF] (mouse cone arrestin promoter) ; U66698 (human cone alpha-transducin promoter) ; U16824 (human rhodopsin promoter).

Gene

Sequence name Sequence

chicken HIOMT (promoter region) cH1

5' TGTGAGTGCCCATTTTTAAAGATG 3' chicken HIOMT (promoter region with mutation) cH1mut

5' TGTGAGTGCCCCTTTTTAAAGATG 3' chicken HIOMT (promoter region) cH2

5' CCTCTGGTCCCATTTCATTGGGCT 3' chicken AANAT (promoter region) cNAT 5' GGATAATCCCCATTTGGTTCCACT 3' chicken rhodopsin (promoter region) cRho 5' TAACGTGTCCCATTTCCCATATCC 3' rat AANAT (intron 1) rNATi 5' CGCCATCGCCCATTTAAAAAGATT 3' mouse cone arrestin (promoter region) mArr 5' CGGTTCTTCCCATTTTGGCTACAT 3' human cone alpha-transducin (promoter region) hTrans 5' ATGATCTGCCCATTTTGGCCTCCC 3' human rhodopsin (promoter region) hRho 5' TTATCATTCCCATTTTACACGAGT 3' In A-D, 32 P-labelled double-stranded oligonucleotides corresponding to putative YY1-binding elements (see Table 1) were analyzed by EMSA. In A, 20 µg of proteins extracted from chicken retina were incubated with putative YY1-binding sites identified in the chicken HIOMT promoter (cH1 and cH2), in the chicken rhodopsin promoter (cRho) and in the chicken AANAT promoter (cNAT). The reactions were carried out in the pcDNA3 plasmids expressing either chicken YY1 (pcDNA3-YY1) or rat Crx (pcDNA3-Crx).

As a negative control, a group of HEK293 cells was cotransfected with pGL3-HIOMT and an empty pCDNA3 plasmid (not shown). In each experimental group, the cells were cotranfected with 1 ng of pRL-SV40, to correct for variations in transfection efficiency. In E, right panel, chicken embryonic retinal cells were transfected with 2 µg of the wild-type promoter-reporter vector pGL3-HIOMT or with 2 µg of pGL3-HIOMT(cH1mut), a pGL3-HIOMT vector

Figure 1 .

 1 Figure 1. Western blot immunodetection of YY1 in chicken tissues and in the developing

Figure 2 .

 2 Figure 2. Immunohistochemical detection of YY1 in the retina of dark-adapted

Figure 3 :

 3 Figure 3: Immunohistochemical detection of YY1 in the retina of light-adapted chickens.

Figure 4 .

 4 Figure 4. Analysis of putative YY1-regulatory elements, by EMSA and transient transfections. In A-D, 32 P-labelled double-stranded oligonucleotides corresponding to

  carrying a mutation of the cH1 sequence that abolishes YY1-binding (see panel B). The retinal cells were cotranfected with 200 ng of pRL-SV40, to correct for variations in transfection efficiency. In HEK293 and retinal cells, firefly and Renilla luciferase activities were assayed 24h post-transfection. The ratios of firefly/Renilla luciferase activities were calculated and were expressed relative to (pGL3-HIOMT+ empty pcDNA3) in HEK293, and to pGL3-HIOMT in retinal cells. Histograms represent the mean ± S.E.M. (n=3 in HEK cells ; n=12 in retinal cells). Overexpression of YY1 did not activate the HIOMT promoter in HEK293 cells, while overexpression of Crx did (p<0.05 at the Student's t-test). In retinal cells the mutation of the YY1-binding site decreased the HIOMT promoter activity by 70% (p<0.005 at the Student's t-test).

Figure 1 Figure 4

 14 Figure 1

ACKNOWLEDGEMENTS

This work was supported by the CNRS (UMR 6187), Fondation Langlois and Retina France.

We would like to thank Dr. A.C. Balandre for her precious help on the cultures of HEK293 cells. We would like to thank L. Cousin for her technical help at multiple steps of this work.

We appreciate the technical assistance of the University facilities: Service Interdisciplinaire de Microscopie et d'Imagerie Scientifique (Dr. E. Béré), Service Commun de Séquençage (D.

Guyonnet) and Service Commun de Publication Assistée par Ordinateur (J. Habrioux).

absence (-) or in the presence (+) of a 200-fold molar excess of unlabelled DNA competitor homologous to the labelled probe. Only cH1 shows a retarded band that is specifically displaced by the unlabelled competitor (arrow). In B, the cH1 labelled probe was incubated with 20 µg of proteins from chicken retina, in the absence (-) or in the presence of increasing amounts (50-, 100-, 200-or 500-fold molar excesses) of unlabelled DNA competitor homologous to the labelled probe (cH1) or carrying a point mutation (cH1mut: CCATTT → CCCTTT). The cH1mut DNA was also used as a 32 P-labelled probe, in the absence or presence of the homologous unlabelled competitor (200-fold molar excess). The retinal protein that binds cH1 can be saturated by unlabelled cH1 DNA, but does not bind unlabelled or labelled cH1mut. In C, retinal proteins were incubated with 32 P-labelled cH1, in the presence of the anti-YY1 antibody (-YY1) or in the presence of normal rabbit IgG (ctrl-Ig). The binding of the retinal protein to cH1 is inhibited by the anti-YY1 antibody, but not by control immunoglobulins. A retarded band is also observed when the cH1 probe is incubated with in vitro-expressed chicken YY1 (arrow). This band is absent when the cH1 probe is incubated with the reticulocyte lysate alone (RL). In D, 32 P-labelled probes corresponding to putative YY1-binding sites identified in intron1 of the rat AANAT gene (rNATi) and in the human transducin promoter (hTrans) were tested by EMSA. When rNATi was incubated with proteins from rat retina and when hTrans was incubated with proteins from human Y79 retinoblastoma cells, retarded bands that were recognized by the anti-YY1 antibody but not by control immunoglobulins were observed. The bands pointed out by the long arrow have the same electrophoretic mobility as the one observed with cH1. A second band of higher mobility that is also recognized by the anti-YY1 antibody is observed with the rNATi probe (short arrow). This band might correspond to a truncated form of YY1 still containing the Cterminal domain. In E, left panel, HEK293 cells were cotransfected with 50 ng of a promoterreporter vector containing 3kb of chicken HIOMT promoter (pGL3-HIOMT) and 2 µg of