Background Microarray is a high-throughput technology to study expression of thousands of genes in parallel. A critical aspect of microarray production is the design aimed at space optimization while maximizing the number of gene probes and their replicates to be spotted.
Background
Microarray is a high-throughput technology to study expression of thousands of genes in parallel. A critical aspect of microarray production is the design aimed at space optimization while maximizing the number of gene probes and their replicates to be spotted.Results We have developed a software called 'ArrayD' that offers various alternative design solutions for an array given a set of user requirements. The user feeds the following inputs: type of source plates to be used, number of gene probes to be printed, number of replicates and number of pins to be used for printing. The solutions are stored in a text file. The choice of a design solution to be used will be governed by the spotting chemistry to be used and the accuracy of the robot.
Results
We have developed a software called 'ArrayD' that offers various alternative design solutions for an array given a set of user requirements. The user feeds the following inputs: type of source plates to be used, number of gene probes to be printed, number of replicates and number of pins to be used for printing. The solutions are stored in a text file. The choice of a design solution to be used will be governed by the spotting chemistry to be used and the accuracy of the robot.Conclusions ArrayD is a software for standard cartesian robots. The software aids users in preparing a judicious and elegant design. ArrayD is universally applicable and is available at  .
Conclusions
ArrayD is a software for standard cartesian robots. The software aids users in preparing a judicious and elegant design. ArrayD is universally applicable and is available at  .

Background Microarray is a popularly used high-throughput technology to investigate gene expression of thousands of genes simultaneously at the level of mRNA. Ever since the development of this technology [ 1 - 3 ], transcriptional profiling at the genomic level has been employed to address numerous issues in biology and in medicine [ 4 - 8 ]. It is likely that microarrays will continue to be used to explore various biological phenomena. The basic underlying principle involves spotting DNA fragments either derived from polymerase chain reaction or preparation of plasmids or oligonucleotides at high density (~10,000–25,000 spots on a glass slide of 25 mm × 75 mm) representing the probes of the genes under study. The surface on which the DNA fragments or oligonucleotides are spotted is usually glass slides coated with poly-L-lysine or amino alkyl silane that serve to improve adherence of DNA to the surface. Uniform spotting at high density requires robotic operation and a variety of robots are now available for spotting [ 9 ]. The robots employed for the preparation of microarrays are of the cartesian type with movement in x-y-z direction. A critical aspect of microarray production is the design considering space optimization to produce high-density arrays for a given set of samples and replicates. The softwares generally supplied with robotic spotters translate user input parameters into a set of instructions in robot language for printing arrays. These softwares do not offer design capabilities in which spotting parameters and grid configurations can be chosen for a given set of samples and replicates. Presently various solutions have to be derived manually in most academic laboratories. We have developed a user-friendly software 'ArrayD' that can be used by experts and novice alike to fill this gap to simplify and aid in rapid design. ArrayD offers a variety of design solutions given a set of requirements: Number of gene probes, number of replicates, and the source plate (384 well or 96 well). Because the algorithm implemented in ArrayD is inherently simple and uses fundamental principles of robot operation, the design solutions offered by ArrayD are universally applicable to any system. The choice of a design solution would be governed by the spotting chemistry and the humidity used in addition to elegant appearance. The hallmark of ArrayD is its overall simplicity and the variety of alternative designs it offers for users to decide on choosing the appropriate spotting parameters. The multiple design solutions offered by ArrayD provides a wide range of arrays from compact to loosely spaced spots as well as convenient grid patterning, which can be user selected for printing. Implementation ArrayD program is developed in C and can be compiled and operated on UNIX V5.1, IRIX 5.1 and Red Hat Linux 7.0 (or higher) operating systems. A companion computer program ArraySolution was developed in Perl (Practical Extraction Report Language) version 5.6.1 and can be implemented on any UNIX or Linux operating system. Inputs to be defined for ArrayD (a) Type of source plate to be used The input parameter toggles between either 96 well or 384 well plate. (b) Number of gene probes to be printed The number of gene probes including positive and negative controls and blanks. (c) Number of replicates The number of replicates of each gene probe. Although the most common pattern chosen is duplicates, users can choose any number of replicates. Replicates are assumed to be printed in the Y axis. (d) Number of pins to be used for printing This parameter relates to time taken for printing the slides and the number of spots arrayed per slide. The number of pins in X-axis and Y-axis need to be specified. The type of pins used is assumed to be stealth pins, which are widely used. It is not necessary to specify pin type for ArrayD. Instead, this aspect is considered in the printing software according to the pin type used for implementing a particular design.
Background
Microarray is a popularly used high-throughput technology to investigate gene expression of thousands of genes simultaneously at the level of mRNA. Ever since the development of this technology [ 1 - 3 ], transcriptional profiling at the genomic level has been employed to address numerous issues in biology and in medicine [ 4 - 8 ]. It is likely that microarrays will continue to be used to explore various biological phenomena. The basic underlying principle involves spotting DNA fragments either derived from polymerase chain reaction or preparation of plasmids or oligonucleotides at high density (~10,000–25,000 spots on a glass slide of 25 mm × 75 mm) representing the probes of the genes under study. The surface on which the DNA fragments or oligonucleotides are spotted is usually glass slides coated with poly-L-lysine or amino alkyl silane that serve to improve adherence of DNA to the surface. Uniform spotting at high density requires robotic operation and a variety of robots are now available for spotting [ 9 ]. The robots employed for the preparation of microarrays are of the cartesian type with movement in x-y-z direction.A critical aspect of microarray production is the design considering space optimization to produce high-density arrays for a given set of samples and replicates. The softwares generally supplied with robotic spotters translate user input parameters into a set of instructions in robot language for printing arrays. These softwares do not offer design capabilities in which spotting parameters and grid configurations can be chosen for a given set of samples and replicates. Presently various solutions have to be derived manually in most academic laboratories. We have developed a user-friendly software 'ArrayD' that can be used by experts and novice alike to fill this gap to simplify and aid in rapid design. ArrayD offers a variety of design solutions given a set of requirements: Number of gene probes, number of replicates, and the source plate (384 well or 96 well). Because the algorithm implemented in ArrayD is inherently simple and uses fundamental principles of robot operation, the design solutions offered by ArrayD are universally applicable to any system. The choice of a design solution would be governed by the spotting chemistry and the humidity used in addition to elegant appearance. The hallmark of ArrayD is its overall simplicity and the variety of alternative designs it offers for users to decide on choosing the appropriate spotting parameters. The multiple design solutions offered by ArrayD provides a wide range of arrays from compact to loosely spaced spots as well as convenient grid patterning, which can be user selected for printing.Implementation ArrayD program is developed in C and can be compiled and operated on UNIX V5.1, IRIX 5.1 and Red Hat Linux 7.0 (or higher) operating systems. A companion computer program ArraySolution was developed in Perl (Practical Extraction Report Language) version 5.6.1 and can be implemented on any UNIX or Linux operating system. Inputs to be defined for ArrayD (a) Type of source plate to be used The input parameter toggles between either 96 well or 384 well plate. (b) Number of gene probes to be printed The number of gene probes including positive and negative controls and blanks. (c) Number of replicates The number of replicates of each gene probe. Although the most common pattern chosen is duplicates, users can choose any number of replicates. Replicates are assumed to be printed in the Y axis. (d) Number of pins to be used for printing This parameter relates to time taken for printing the slides and the number of spots arrayed per slide. The number of pins in X-axis and Y-axis need to be specified. The type of pins used is assumed to be stealth pins, which are widely used. It is not necessary to specify pin type for ArrayD. Instead, this aspect is considered in the printing software according to the pin type used for implementing a particular design.
Implementation
ArrayD program is developed in C and can be compiled and operated on UNIX V5.1, IRIX 5.1 and Red Hat Linux 7.0 (or higher) operating systems. A companion computer program ArraySolution was developed in Perl (Practical Extraction Report Language) version 5.6.1 and can be implemented on any UNIX or Linux operating system.Inputs to be defined for ArrayD (a) Type of source plate to be used The input parameter toggles between either 96 well or 384 well plate. (b) Number of gene probes to be printed The number of gene probes including positive and negative controls and blanks. (c) Number of replicates The number of replicates of each gene probe. Although the most common pattern chosen is duplicates, users can choose any number of replicates. Replicates are assumed to be printed in the Y axis. (d) Number of pins to be used for printing This parameter relates to time taken for printing the slides and the number of spots arrayed per slide. The number of pins in X-axis and Y-axis need to be specified. The type of pins used is assumed to be stealth pins, which are widely used. It is not necessary to specify pin type for ArrayD. Instead, this aspect is considered in the printing software according to the pin type used for implementing a particular design.
Inputs to be defined for ArrayD
(a) Type of source plate to be usedThe input parameter toggles between either 96 well or 384 well plate.(b) Number of gene probes to be printedThe number of gene probes including positive and negative controls and blanks.(c) Number of replicatesThe number of replicates of each gene probe. Although the most common pattern chosen is duplicates, users can choose any number of replicates. Replicates are assumed to be printed in the Y axis.(d) Number of pins to be used for printingThis parameter relates to time taken for printing the slides and the number of spots arrayed per slide. The number of pins in X-axis and Y-axis need to be specified. The type of pins used is assumed to be stealth pins, which are widely used. It is not necessary to specify pin type for ArrayD. Instead, this aspect is considered in the printing software according to the pin type used for implementing a particular design.Results and discussion A general microarray design layout is displayed in Figure  1 . ArrayD accepts standard slide dimensions (25 mm × 75 mm), conceptualizes the spotting area to be 50 mm × 22 mm to provide space for barcode labeling and for appropriate placement of coverslip over the print area. The reference direction of the robot for picking probes from source plate is left → right followed by top → down; the printing direction is top → down followed by left → right. Replicates are considered to be spotted in Y-axis (Figure  1 ). After the user has entered the parameters, the software generates a text file called 'solution.txt' that carries possible alternative array design parameters for the given input. The algorithm implemented in ArrayD is displayed in Figure  2 . The program first validates the input given by the user for appropriate number of pins in each direction and the plate type to be used. For a valid input, ArrayD calculates maximum possible number of super grids in X (or Y) direction based on the coverslip dimensions, pin number in X (or Y) direction and pin-to-pin distance (Figure  2 ). The coverslip dimensions have been set in the program as 50 mm × 22 mm for the longest size coverslip that can be effectively used during hybridization. The pin-to-pin distance is fixed at 4500 microns in the print head for 384 well plates and at 9000 microns for 96 well plates. ArrayD uses a predefined inter-spot distance database. Design solutions of ArrayD encompass various inter-spot distances that would be compatible with different spotting chemistries and conditions of humidity. We have used inter-spot distances of 170 μm, 180 μm, 190 μm, 200 μm, 220 μm, 250 μm and 300 μm. This database can be expanded to incorporate even lower inter-spot distances for use with other spotting chemistries by simple modification. We chose 170 μm as least distance based on several trial experiments in our laboratory using 50% DMSO as spotting solution and SMP3 pin type. In our experience, a minimum inter-spot distance of 200 microns works best with 50% DMSO at 40% – 50% humidity at 25°C. The options for the inter-spot distance currently offered by the software can work successfully for SMP2, SMP2B, SMP2XB, SMP2.5, SMP2.5B, SMP2.5XB, SMP3, SMP3B, SMP3XB, SMP4, SMP4B, SMP4XB, SMP5, SMP5B and SMP5XB stealth pins (See Table  1 ). For each possible super grid configuration, the number of grids in each direction is optimized based on the number of gene probes (samples) input by the user as shown in figure  2 . Design solutions offered by the program Alternative array designs for a given set of input parameters are ranked on the basis of 'Distance area ratio' that describes the area covered by the array for each design. The array design spanning least area is ranked highest. This strategy allows for applying the labeled target sparingly. Subsequently, an easy report in tabular form can be generated by feeding the output data file from ArrayD into the companion Perl program 'ArraySolution.pl', which classifies array solutions into 'Square', 'Rectangle (Horizontal bar)', or 90° rotated 'Rectangle (Vertical column)' based on the geometry of a given design solution. If the number of grids are equal in both the direction we have a 'Square' design. In all other cases we obtain a 'Rectangle' design, which can be either of two types: the long side of the array is parallel to the length (Horizontal) or the width (Vertical) of the slide. The output of ArraySolution is a tab-delimited text file called 'filename.solution' where filename corresponds to the input name of the file carrying design solutions. The tabular report consist of Number of super grids in X – direction, Number of super grids in Y – direction, Number of spots per grid in X – direction, Number of spots per grid in Y – direction, Distance between two spots (in microns), Distance Area ratio and geometry of design (Square or Rectangle). This can aid users to decide on a particular design solution based on space optimization and elegant appearance. An example of a sample run is provided in Figure  3 . The number of gene probes (including controls and blanks) to be spotted using a 2 by 2 pin configuration in X-Y axis is fed as 2304 (Figure  3 ). The gene probes have to be spotted in duplicates so the total number of spots on the slide would be 4608. The program provides 67 different array designs for various inter-spot distances, number of grids and number of spots in each grid. Two examples of different solutions are presented in Figure  4 . In this example, the first solution is ranked highest with inter-spot distance of 170 microns and a 24 × 24 grid pattern with 4 grids in Y axis and 2 grids in X axis. An alternative solution provides a design with a higher inter-spot distance of 200 microns and 18 × 16 grid pattern with 4 grids each in X axis and Y axis. The first solution can be used in conditions when humidity is low and the spotting solution does not absorb moisture and spread after printing. The second solution is more appropriate for printing samples in 50% DMSO. The classification of all design solutions based on the geometries obtained from ArraySolution is displayed in Table 2 [see  Additional file 1 ].
Results and discussion
A general microarray design layout is displayed in Figure  1 . ArrayD accepts standard slide dimensions (25 mm × 75 mm), conceptualizes the spotting area to be 50 mm × 22 mm to provide space for barcode labeling and for appropriate placement of coverslip over the print area.The reference direction of the robot for picking probes from source plate is left → right followed by top → down; the printing direction is top → down followed by left → right. Replicates are considered to be spotted in Y-axis (Figure  1 ). After the user has entered the parameters, the software generates a text file called 'solution.txt' that carries possible alternative array design parameters for the given input. The algorithm implemented in ArrayD is displayed in Figure  2 .The program first validates the input given by the user for appropriate number of pins in each direction and the plate type to be used. For a valid input, ArrayD calculates maximum possible number of super grids in X (or Y) direction based on the coverslip dimensions, pin number in X (or Y) direction and pin-to-pin distance (Figure  2 ). The coverslip dimensions have been set in the program as 50 mm × 22 mm for the longest size coverslip that can be effectively used during hybridization. The pin-to-pin distance is fixed at 4500 microns in the print head for 384 well plates and at 9000 microns for 96 well plates.ArrayD uses a predefined inter-spot distance database. Design solutions of ArrayD encompass various inter-spot distances that would be compatible with different spotting chemistries and conditions of humidity. We have used inter-spot distances of 170 μm, 180 μm, 190 μm, 200 μm, 220 μm, 250 μm and 300 μm. This database can be expanded to incorporate even lower inter-spot distances for use with other spotting chemistries by simple modification. We chose 170 μm as least distance based on several trial experiments in our laboratory using 50% DMSO as spotting solution and SMP3 pin type. In our experience, a minimum inter-spot distance of 200 microns works best with 50% DMSO at 40% – 50% humidity at 25°C.The options for the inter-spot distance currently offered by the software can work successfully for SMP2, SMP2B, SMP2XB, SMP2.5, SMP2.5B, SMP2.5XB, SMP3, SMP3B, SMP3XB, SMP4, SMP4B, SMP4XB, SMP5, SMP5B and SMP5XB stealth pins (See Table  1 ). For each possible super grid configuration, the number of grids in each direction is optimized based on the number of gene probes (samples) input by the user as shown in figure  2 .Design solutions offered by the program Alternative array designs for a given set of input parameters are ranked on the basis of 'Distance area ratio' that describes the area covered by the array for each design. The array design spanning least area is ranked highest. This strategy allows for applying the labeled target sparingly. Subsequently, an easy report in tabular form can be generated by feeding the output data file from ArrayD into the companion Perl program 'ArraySolution.pl', which classifies array solutions into 'Square', 'Rectangle (Horizontal bar)', or 90° rotated 'Rectangle (Vertical column)' based on the geometry of a given design solution. If the number of grids are equal in both the direction we have a 'Square' design. In all other cases we obtain a 'Rectangle' design, which can be either of two types: the long side of the array is parallel to the length (Horizontal) or the width (Vertical) of the slide. The output of ArraySolution is a tab-delimited text file called 'filename.solution' where filename corresponds to the input name of the file carrying design solutions. The tabular report consist of Number of super grids in X – direction, Number of super grids in Y – direction, Number of spots per grid in X – direction, Number of spots per grid in Y – direction, Distance between two spots (in microns), Distance Area ratio and geometry of design (Square or Rectangle). This can aid users to decide on a particular design solution based on space optimization and elegant appearance. An example of a sample run is provided in Figure  3 . The number of gene probes (including controls and blanks) to be spotted using a 2 by 2 pin configuration in X-Y axis is fed as 2304 (Figure  3 ). The gene probes have to be spotted in duplicates so the total number of spots on the slide would be 4608. The program provides 67 different array designs for various inter-spot distances, number of grids and number of spots in each grid. Two examples of different solutions are presented in Figure  4 . In this example, the first solution is ranked highest with inter-spot distance of 170 microns and a 24 × 24 grid pattern with 4 grids in Y axis and 2 grids in X axis. An alternative solution provides a design with a higher inter-spot distance of 200 microns and 18 × 16 grid pattern with 4 grids each in X axis and Y axis. The first solution can be used in conditions when humidity is low and the spotting solution does not absorb moisture and spread after printing. The second solution is more appropriate for printing samples in 50% DMSO. The classification of all design solutions based on the geometries obtained from ArraySolution is displayed in Table 2 [see  Additional file 1 ].
Design solutions offered by the program
Alternative array designs for a given set of input parameters are ranked on the basis of 'Distance area ratio' that describes the area covered by the array for each design. The array design spanning least area is ranked highest. This strategy allows for applying the labeled target sparingly. Subsequently, an easy report in tabular form can be generated by feeding the output data file from ArrayD into the companion Perl program 'ArraySolution.pl', which classifies array solutions into 'Square', 'Rectangle (Horizontal bar)', or 90° rotated 'Rectangle (Vertical column)' based on the geometry of a given design solution. If the number of grids are equal in both the direction we have a 'Square' design. In all other cases we obtain a 'Rectangle' design, which can be either of two types: the long side of the array is parallel to the length (Horizontal) or the width (Vertical) of the slide. The output of ArraySolution is a tab-delimited text file called 'filename.solution' where filename corresponds to the input name of the file carrying design solutions. The tabular report consist of Number of super grids in X – direction, Number of super grids in Y – direction, Number of spots per grid in X – direction, Number of spots per grid in Y – direction, Distance between two spots (in microns), Distance Area ratio and geometry of design (Square or Rectangle). This can aid users to decide on a particular design solution based on space optimization and elegant appearance.An example of a sample run is provided in Figure  3 . The number of gene probes (including controls and blanks) to be spotted using a 2 by 2 pin configuration in X-Y axis is fed as 2304 (Figure  3 ). The gene probes have to be spotted in duplicates so the total number of spots on the slide would be 4608. The program provides 67 different array designs for various inter-spot distances, number of grids and number of spots in each grid. Two examples of different solutions are presented in Figure  4 . In this example, the first solution is ranked highest with inter-spot distance of 170 microns and a 24 × 24 grid pattern with 4 grids in Y axis and 2 grids in X axis. An alternative solution provides a design with a higher inter-spot distance of 200 microns and 18 × 16 grid pattern with 4 grids each in X axis and Y axis. The first solution can be used in conditions when humidity is low and the spotting solution does not absorb moisture and spread after printing. The second solution is more appropriate for printing samples in 50% DMSO. The classification of all design solutions based on the geometries obtained from ArraySolution is displayed in Table 2 [see  Additional file 1 ].Conclusion We have developed a simple and rapid software ArrayD that offers various design solutions of designing microarrays for a specific set of user-defined requirements.
Conclusion
We have developed a simple and rapid software ArrayD that offers various design solutions of designing microarrays for a specific set of user-defined requirements.Availability and requirements The source code and the executable file for ArrayD and ArraySolution programs are freely available and can be downloaded from our website [ 11 ]. The source code can be compiled and executed on Unix v 5.1, or IRIX v 5.1 or Red Hat Linux v 7.0 (or higher). The executable files can be downloaded for Windows platform (Windows 98/NT/XP/2000). Further information can be requested by sending e-mail to  ramu@igib.res.in  or  ramucbt@yahoo.com .
Availability and requirements
The source code and the executable file for ArrayD and ArraySolution programs are freely available and can be downloaded from our website [ 11 ]. The source code can be compiled and executed on Unix v 5.1, or IRIX v 5.1 or Red Hat Linux v 7.0 (or higher). The executable files can be downloaded for Windows platform (Windows 98/NT/XP/2000). Further information can be requested by sending e-mail to  ramu@igib.res.in  or  ramucbt@yahoo.com .Authors' contributions AS explained the operational details to software expert, did the experimentation, maintenance and testing of the software. GPS did the basic software writing and implementation of 'ArrayD'. VKS prepared the code for the companion program 'ArraySolution' to classify the solutions on the basis of design geometry. SR is the Group Leader generating demand, sourcing and linking people, explaining the concepts, testing, critical examination, presentation of data, providing salary through grants.
Authors' contributions
AS explained the operational details to software expert, did the experimentation, maintenance and testing of the software. GPS did the basic software writing and implementation of 'ArrayD'. VKS prepared the code for the companion program 'ArraySolution' to classify the solutions on the basis of design geometry. SR is the Group Leader generating demand, sourcing and linking people, explaining the concepts, testing, critical examination, presentation of data, providing salary through grants.Supplementary Material Additional File 1 Report generated by ArraySolution. A total of 67 different design solutions were classified into three categories of Square, Rectangle (Horizontal) and Rectangle (Vertical) as mentioned in text. The detailed design parameters including number of supergrids in X and Y direction, number of spots per grid in X and Y direction, distance between the spots, distance area ratio and geometry of design are provided for preparing elegant microarrays. Click here for file
Supplementary Material
Additional File 1
Report generated by ArraySolution. A total of 67 different design solutions were classified into three categories of Square, Rectangle (Horizontal) and Rectangle (Vertical) as mentioned in text. The detailed design parameters including number of supergrids in X and Y direction, number of spots per grid in X and Y direction, distance between the spots, distance area ratio and geometry of design are provided for preparing elegant microarrays.Click here for file


Background Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications.  MBD2/3  is the only candidate gene in the  Drosophila  genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the  N ucleosome  R emodelling and  D eacetylase (NuRD) complex, respectively. An association of  Drosophila  MBD2/3 with the  Drosophila  NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.
Background
Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications.  MBD2/3  is the only candidate gene in the  Drosophila  genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the  N ucleosome  R emodelling and  D eacetylase (NuRD) complex, respectively. An association of  Drosophila  MBD2/3 with the  Drosophila  NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.MBD2/3DrosophilaDrosophilaDrosophilaResults The two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the  Drosophila  NuRD complex.
Results
The two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the  Drosophila  NuRD complex.DrosophilaConclusion Our data provide novel insights into the association between  Drosophila  MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the  Drosophila  NuRD complex.
Conclusion
Our data provide novel insights into the association between  Drosophila  MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the  Drosophila  NuRD complex.DrosophilaDrosophila

Background Methyl-DNA binding proteins are connecting DNA methylation to transcriptional silencing [ 1 - 4 ]. Up to now, six methyl-DNA binding proteins could be identified in vertebrates [ 5 ]. MeCP2, MBD2 and MBD3 can be found in large chromatin complexes containing histone deacetylase activity [ 1 , 6 , 4 , 3 ] whereas MBD4 is involved in DNA mismatch-repair [ 7 ]. MBD1 has been shown to repress transcription in cell culture [ 8 ] and recruits the histone H3-K9 methyltransferase SETDB1 to the chromatin assembly factor CAF-1 during S phase [ 9 ]. MBD2, which can bind methylated DNA [ 6 ], is a transcriptional repressor recruiting a  Nu cleosome  R emodelling and  D eacetylase complex (NuRD) to methylated CpG dinucleotides [ 6 , 3 ], whereas mammalian MBD3, which is not able to bind methylated DNA [ 10 ] is an integral component of NuRD [ 3 ]. Kaiso, a transcriptional repressor protein, can bind directly to CpG methylated DNA even though it lacks a conserved methyl-DNA binding domain [ 11 ]. Kaiso is a component of a subpopulation of MeCP1 complexes that lack MBD2 [ 11 ]. The  Drosophila  gene  MBD2/3  encodes a protein, which shares high homology to mammalian MBD2 and MBD3 [ 12 , 4 ]. Due to differential splicing,  Drosophila MBD2/3  is expressed in two isoforms, the smaller one is lacking part of the putative methyl-DNA binding domain [ 12 - 15 ]. The large isoform is expressed during early development, whereas the small isoform can only be detected during late embryogenesis [ 14 , 15 ]. In insect cells expressing only the small MBD2/3 isoform, this protein was found to be associated with components of the  Drosophila  NuRD complex [ 12 - 14 ]. Moreover, this protein could repress transcription effectively in transfected  Drosophila  cells [ 13 , 14 ]. The NuRD complexes of vertebrates are a heterogeneous group of complexes containing both histone deacetylase and nucleosome remodelling activities [ 3 ]. NuRD complexes comprise at least seven proteins. The ATP- dependent nucleosome-remodelling activity is mediated by MI-2, which contains a SWI2/SNF2-type helicase/ATPase domain, two chromodomains and two PHD fingers [ 16 ]. The related MTA1, MTA2 and MTA3 proteins have been found in various complex preparations [ 17 , 4 , 3 , 18 ]. MTA1 was originally identified as being overexpressed in metastatic carcinomas [ 19 ]. The histone deacetylases HDAC1 and HDAC2 and the two histone binding proteins RbAp46 and RbAp48 form the histone deacetylase core of NuRD complexes. Finally, as mentioned above, mammalian MBD3 is an integral component of at least some NuRD complexes [ 3 ]. Strikingly, the  Drosophila  genome contains clear homologues for all verterbrate NuRD proteins. Recombinant  Drosophila  MI-2 was shown to have ATPase and nucleosome mobilization properties [ 20 ]. In  Drosophila  the HDAC gene  Rpd3  is important for segmentation of the embryo [ 21 ].  Drosophila  p55, a WD-40 protein, is homologous to the histone deacetylase-associated proteins RbAp46 and RbAp48 [ 22 ]. Finally,  Drosophila  MTA-like displays extensive homology to the vertebrate MTA proteins [ 23 ]. The strong conservation between vertebrate NuRD complexes and the  Drosophila  NuRD complex implies a conserved function during the animal development. In previous studies cell lines were analysed that express only the small isoform of MBD2/3, lacking part of the putative methyl-DNA binding domain and a  Drosophila  specific domain. In order to analyse isoform-specific differences of MBD2/3 and their ability to bind NuRD proteins, we now extend our analysis to both isoforms in  Drosophila  embryos.
Background
Methyl-DNA binding proteins are connecting DNA methylation to transcriptional silencing [ 1 - 4 ]. Up to now, six methyl-DNA binding proteins could be identified in vertebrates [ 5 ]. MeCP2, MBD2 and MBD3 can be found in large chromatin complexes containing histone deacetylase activity [ 1 , 6 , 4 , 3 ] whereas MBD4 is involved in DNA mismatch-repair [ 7 ]. MBD1 has been shown to repress transcription in cell culture [ 8 ] and recruits the histone H3-K9 methyltransferase SETDB1 to the chromatin assembly factor CAF-1 during S phase [ 9 ]. MBD2, which can bind methylated DNA [ 6 ], is a transcriptional repressor recruiting a  Nu cleosome  R emodelling and  D eacetylase complex (NuRD) to methylated CpG dinucleotides [ 6 , 3 ], whereas mammalian MBD3, which is not able to bind methylated DNA [ 10 ] is an integral component of NuRD [ 3 ]. Kaiso, a transcriptional repressor protein, can bind directly to CpG methylated DNA even though it lacks a conserved methyl-DNA binding domain [ 11 ]. Kaiso is a component of a subpopulation of MeCP1 complexes that lack MBD2 [ 11 ].The  Drosophila  gene  MBD2/3  encodes a protein, which shares high homology to mammalian MBD2 and MBD3 [ 12 , 4 ]. Due to differential splicing,  Drosophila MBD2/3  is expressed in two isoforms, the smaller one is lacking part of the putative methyl-DNA binding domain [ 12 - 15 ]. The large isoform is expressed during early development, whereas the small isoform can only be detected during late embryogenesis [ 14 , 15 ]. In insect cells expressing only the small MBD2/3 isoform, this protein was found to be associated with components of the  Drosophila  NuRD complex [ 12 - 14 ]. Moreover, this protein could repress transcription effectively in transfected  Drosophila  cells [ 13 , 14 ].DrosophilaMBD2/3Drosophila MBD2/3DrosophilaDrosophilaThe NuRD complexes of vertebrates are a heterogeneous group of complexes containing both histone deacetylase and nucleosome remodelling activities [ 3 ]. NuRD complexes comprise at least seven proteins. The ATP- dependent nucleosome-remodelling activity is mediated by MI-2, which contains a SWI2/SNF2-type helicase/ATPase domain, two chromodomains and two PHD fingers [ 16 ]. The related MTA1, MTA2 and MTA3 proteins have been found in various complex preparations [ 17 , 4 , 3 , 18 ]. MTA1 was originally identified as being overexpressed in metastatic carcinomas [ 19 ]. The histone deacetylases HDAC1 and HDAC2 and the two histone binding proteins RbAp46 and RbAp48 form the histone deacetylase core of NuRD complexes. Finally, as mentioned above, mammalian MBD3 is an integral component of at least some NuRD complexes [ 3 ].Strikingly, the  Drosophila  genome contains clear homologues for all verterbrate NuRD proteins. Recombinant  Drosophila  MI-2 was shown to have ATPase and nucleosome mobilization properties [ 20 ]. In  Drosophila  the HDAC gene  Rpd3  is important for segmentation of the embryo [ 21 ].  Drosophila  p55, a WD-40 protein, is homologous to the histone deacetylase-associated proteins RbAp46 and RbAp48 [ 22 ]. Finally,  Drosophila  MTA-like displays extensive homology to the vertebrate MTA proteins [ 23 ].DrosophilaDrosophilaDrosophilaRpd3DrosophilaDrosophilaThe strong conservation between vertebrate NuRD complexes and the  Drosophila  NuRD complex implies a conserved function during the animal development. In previous studies cell lines were analysed that express only the small isoform of MBD2/3, lacking part of the putative methyl-DNA binding domain and a  Drosophila  specific domain. In order to analyse isoform-specific differences of MBD2/3 and their ability to bind NuRD proteins, we now extend our analysis to both isoforms in  Drosophila  embryos.DrosophilaDrosophilaDrosophilaResults MBD2/3 is associated with  Drosophila  NuRD proteins An association of  Drosophila  MBD2/3 with the NuRD complex has been suggested previously [ 14 , 12 ]. However, these experiments were done in vitro or with protein extracts from tissue culture cells that express only the small isoform of MBD2/3. In addition, only a limited number of NuRD proteins were analysed. To confirm the association between MBD2/3 and NuRD in  Drosophila  embryos we size-fractionated nuclear protein extracts from 2–4 h or 0–12 h wild type embryos, respectively, by Superose 6 gel filtration chromatography. Proteins from individual fractions were then separated by standard SDS-PAGE and analysed by Western blotting using antibodies directed against known homologous subunits of the NuRD complex. MI-2 and MTA-like, which are homologues of subunits that are specific for vertebrate NuRD complexes [ 24 , 3 ] cofractionated with an apparent molecular weight of 1 MDa when extracts derived from early embryos (2–4 h) were applied (Fig.  1A , fractions 19 to 21). When extracts of late embryos (0–12 h) were analysed, MI-2 and MTA-like were detected in fractions 15 to 19, which correspond to an apparent molecular weight of approx. 2 MDa (Fig.  1B ). This result indicates that the formation of NuRD is developmentally regulated during embryogenesis. Additionally, we found a precise cofractionation of the large isoform of MBD2/3 (Fig.  1 , MBD2/3li) with MI-2 and MTA-like in both cases. The small MBD2/3 isoform (Fig.  1B , MBD2/3si) present in extracts derived from late embryos likewise cofractionated with MI-2 and MTA-like. This suggests a close association of both MBD2/3 isoforms with the  Drosophila  NuRD complex. The RPD3 and p55 proteins also cofractionated with MBD2/3 isoforms, but they were also found in fractions of lower molecular weight (Fig.  1 ). This is in agreement with the fact that both RPD3 and p55 are also components of other chromatin related complexes [ 25 , 26 ]. Figure 1 Cofractionation of  Drosophila  NuRD homologues in embryonic protein extracts  Nuclear protein extracts were prepared from (A) 2–4 h and (B) 0–12 h old embryos, respectively and size-fractionated by FPLC using a Superose 6 column. Proteins from selected fractions were then separated by SDS gel electrophoresis and analysed by Western blotting. (A) During early embryogenesis the long isoform of MBD2/3 (MBD2/3li) cofractionated with MI-2 and MTA-like in fractions 19 to 21 (indicated in red), which correspond to a molecular weight of approx. 1 MDa. The small isoform of MBD2/3 is not expressed at this stage of embryogenesis. (B) During later stages of embryogenesis both isoforms of MBD2/3 cofractionated with MI-2 and MTA-like in fractions 15 to 19 (indicated in red), which correspond to a molecular weight of approx. 2 MDa. The size of marker proteins is shown left and on top, IN indicates input protein. Interactions between MBD2/3 and NuRD homologues To analyse the association between MBD2/3 and the NuRD complex in more detail and to identify direct NuRD interaction partner(s) of MBD2/3 we first used a GST-pulldown assay. In order to eliminate unspecific interactions with NuRD proteins, we established highly stringent assay conditions. This was achieved by assessing the interactions between radioactively labeled SV40 large T antigen (Fig.  2A , large T) and a number of control GST fusion proteins. When we used an incubation and washing buffer with high salt and detergent concentrations, significant amounts of SV40 large T were precipitated by p53 only (Fig.  2A ), which is known to be a strong interactor of the large T antigen [ 27 ]. Residual binding detected with some of the other proteins (Fig.  2A ) was considered background. Figure 2 Interactions between MBD2/3 and NuRD homologues in a GST-pulldown assay  (A) Analysis of interactions between radioactively labelled SV40 large T antigen (large T) and a number of control GST fusion proteins under stringent buffer conditions. Significant amounts of SV40 large T were only precipitated by p53. The faint bands seen with some of the other proteins were considered background. (B) GST-MBD2/3 fusion proteins for long and small isoforms (MBD2/3li, MBD2/3si, respectively) efficiently precipitated radioactively labelled MBD2/3 long isoform and p55 (solid arrowhead). Similarly, MI-2 protein could be precipitated by the small GST-MBD2/3 isoform (solid arrowhead). No interaction could be observed between long MBD2/3 or small MBD2/3 isoforms and MTA-like or RPD3 (open arrowheads). (C) A MBD2/3-specific antibody immunoprecipitates p55, but not GAGA factor from embryonic nuclear extracts. No proteins were detectable in control immunoprecipitations with a myc-specific antibody. (D) Size-fractionation of baculovirus-expressed MBD2/3 long isoform and RPD3. Baculovirus-expressed MBD2/3li elutes in fractions that significantly exceed the calculated molecular weight of the protein (36 kDa), thus indicating that MBD2/3li efficiently multimerizes in solution. This effect appeared to be specific for MBD2/3 and was not observed with baculovirus-expressed RPD3 protein (58kDa). The size of marker proteins is shown left and on top, IN indicates input protein. We then performed the MBD2/3-NuRD interaction assay using these conditions and observed strong interactions only between the long and small MBD2/3 isoforms (Fig.  2B , MBD2/3li and MBD2/3si, respectively) and p55. To confirm the association between MBD2/3 and p55 in  Drosophila  embryos we immunoprecipitated nuclear extracts with MBD2/3-specific antibodies and with myc-specific control antibodies. Precipitates were analysed by Western blot for the presence of p55 and GAGA factor. This revealed a specific association of MBD2/3 with the  Drosophila  NuRD homologue p55, but not with the unrelated GAGA factor (Fig.  2C ). A weaker interaction could also be detected between the small MBD2/3 isoform and MI-2 (Fig.  2B ). Neither RPD3 nor MTA-like binding exceeded the background level defined by our pilot experiment with SV40 large T protein (Fig.  2B ). Our results thus identified p55 and MI-2 as the direct interaction partners of MBD2/3 in the NuRD complex. In addition, our results from the GST-pulldown assay indicate that  Drosophila  MBD2/3 forms dimers or multimers, similar to mammalian MBD2 and MBD3 [ 28 ]. To discriminate between the latter two possibilities we expressed a recombinant long MBD2/3 isoform and RPD3 in insect cells using a baculovirus expression system. Extracts from infected cells were subjected to Superdex 200 gel filtration and fractions were analysed by Western blot. The 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 exists as monomers or dimers in solution (Fig.  2D ). In contrast, the 36 kDa MBD2/3 isoform showed a strikingly different elution profile (Fig.  2D ). The long MBD2/3 isoform eluted in a broad peak ranging from more than 158 kDa up to high molecular weight fractions (>400 kDa). No MBD2/3 was detected in fractions corresponding to the expected size of MBD2/3 monomers or dimers. This result is consistent with our earlier observation that MBD2/3 forms distinct aggregates in embryonic nuclei [ 15 ] and suggests that the protein efficiently oligomerises to form high-molecular weight complexes in solution. To confirm these interactions in an independent set of experiments we also analysed the association between MBD2/3 isoforms and the NuRD proteins in a yeast two-hybrid assay. In agreement with our previous results, growth on highly selective (Fig.  3A , panel H) plates was only observed upon co-expression of the MBD2/3 small isoform and either MI-2, MBD2/3 long isoform or p55, and upon co-expression of both MBD2/3 isoforms. These interactions were confirmed by X-Gal staining of filter-lifted yeast colonies (Fig.  3A , panel X). In these assays the two MBD2/3 isoforms differed only with regard to MI-2 binding (Fig.  3A ). However, differential binding could be eliminated by a slight reduction in the stringency of the assay (Fig.  3A , panel M), which suggested that both isoforms of MBD2/3 are capable of interacting with MI-2. Consistent with our GST-pulldown assays no interaction could be detected between the MBD2/3 long isoform and RPD3 or MTA-like (Fig.  3A ). Figure 3 Interactions between MBD2/3 and NuRD homologues in a yeast two-hybrid assay  (A) An MBD2/3 long isoform bait efficiently transactivated reporter gene transcription in yeast expressing p55 or MBD2/3 small isoform prey constructs, respectively. A weaker transactivation could be seen upon expression of a MI-2 prey construct (panel M). An MBD2/3 small isoform bait transactivated reporter gene transcription co-expressing a MI-2 prey also under high-stringent conditions (panel H). No transactivation could be observed with MTA-like or RPD3 preys. N shows growth on non-selective plates, M on medium-stringency plates and H on high-stringency plates. X indicates X-gal staining of colonies grown on non-selective plates. (B and C) Delineation of interaction domains in a yeast two-hybrid assay. The methyl-DNA binding domain (MBD) is highlighted in red, the  Drosophila  specific domain (DSD) in green and the C-terminal coiled-coil domain (cc) in yellow. (B) The C-terminal region of MBD2/3 interacts with p55. The full-length p55 construct was used as a bait and various deletion mutants of MBD2/3 were used as preys. This identified the region between amino acids 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain. (C) Deletion of the coiled-coil domain abolishes interactions between MBD2/3 small isoform and MI-2 under high stringent conditions. Next we sought to delineate the domains that mediate the association between the MBD2/3 isoforms and their interacting proteins. To this end, we generated several MBD2/3 deletion mutants and tested their interaction with other proteins in a yeast two-hybrid assay. In a first series of experiments we tested the MBD2/3 mutants for their ability to interact with p55. This identified the region between residues 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain (Fig.  3B ). We also delineated the domain that mediates the interaction with MI-2. Our experiments revealed a strongly reduced interaction between a MBD2/3 small isoform derivative lacking the coiled-coil domain and MI-2 (Fig.  3C ). It has been shown previously that vertebrate MBD2 and MBD3 form homo- and heterodimers via their N-terminal MBD domain and their C-terminal coiled-coil like sequences [ 28 ]. Our findings might reflect a direct interaction between the coiled-coil domain and MI-2 or a requirement for efficient MBD2/3 multimerization for MI-2 interaction. In conclusion, our results identify distinct regions in the MBD2/3 protein that mediate protein-protein interactions with other NuRD proteins. In order to analyse the architecture of the NuRD complex, we performed a detailed yeast two-hybrid analysis including additional bait and prey constructs for NuRD proteins. Bait constructs for MI-2, RPD3, p55 and both MBD2/3 isoforms and prey constructs for MTA-like, RPD3, p55, and both MBD2/3 isoforms were co-transformed in all possible combinations and transformation was confirmed by growth on non-selective plates (Fig.  4A ). Colonies were then replicated onto highly selective plates that allow growth only upon interaction between the two expressed proteins (Fig.  4B ). Finally, we performed a filter lift assay followed by X-Gal staining to confirm the interactions (Fig.  4C ). In addition to the results presented above, we found strong interactions between p55 and all NuRD proteins. We also observed a homotypic interaction for p55 but no homotypic interaction for RPD3. To confirm the specificity of the interactions, we tested most of the constructs reciprocally by changing the bait and prey vectors for expression of NuRD proteins and MBD2/3 isoforms. This data allows the establishment of a more detailed model of the NuRD complex. Fig.  4D  summarizes the data from all experiments. p55 interacted with MI-2, MTA-like, RPD3, and both MBD2/3 isoforms. The small isoform of MBD2/3 interacted with MI-2 as well as with the large MBD2/3 isoform. Homodimerization of p55 and MBD2/3 is not shown in the model. Figure 4 Analysis of the architecure of NuRD  (A) Yeast colonies growing on non-selective plates carrying bait (left panel) and prey (top panel) NuRD and MBD2/3 constructs. (B) Replica plate with high-selective medium. Only yeast colonies with constructs encoding interacting proteins are able to grow. MBD2/3li Δ59-339  represents a truncated MBD2/3li construct that was used to confirm the specificity of the interaction between p55 and MBD2/3. (C) X-Gal staining of yeast colonies to confirm interactions. (D) Schematic illustration of protein-protein interactions in the NuRD complex. Homodimerization of p55 and MBD2/3 are not shown.
Results
MBD2/3 is associated with  Drosophila  NuRD proteins An association of  Drosophila  MBD2/3 with the NuRD complex has been suggested previously [ 14 , 12 ]. However, these experiments were done in vitro or with protein extracts from tissue culture cells that express only the small isoform of MBD2/3. In addition, only a limited number of NuRD proteins were analysed. To confirm the association between MBD2/3 and NuRD in  Drosophila  embryos we size-fractionated nuclear protein extracts from 2–4 h or 0–12 h wild type embryos, respectively, by Superose 6 gel filtration chromatography. Proteins from individual fractions were then separated by standard SDS-PAGE and analysed by Western blotting using antibodies directed against known homologous subunits of the NuRD complex. MI-2 and MTA-like, which are homologues of subunits that are specific for vertebrate NuRD complexes [ 24 , 3 ] cofractionated with an apparent molecular weight of 1 MDa when extracts derived from early embryos (2–4 h) were applied (Fig.  1A , fractions 19 to 21). When extracts of late embryos (0–12 h) were analysed, MI-2 and MTA-like were detected in fractions 15 to 19, which correspond to an apparent molecular weight of approx. 2 MDa (Fig.  1B ). This result indicates that the formation of NuRD is developmentally regulated during embryogenesis. Additionally, we found a precise cofractionation of the large isoform of MBD2/3 (Fig.  1 , MBD2/3li) with MI-2 and MTA-like in both cases. The small MBD2/3 isoform (Fig.  1B , MBD2/3si) present in extracts derived from late embryos likewise cofractionated with MI-2 and MTA-like. This suggests a close association of both MBD2/3 isoforms with the  Drosophila  NuRD complex. The RPD3 and p55 proteins also cofractionated with MBD2/3 isoforms, but they were also found in fractions of lower molecular weight (Fig.  1 ). This is in agreement with the fact that both RPD3 and p55 are also components of other chromatin related complexes [ 25 , 26 ]. Figure 1 Cofractionation of  Drosophila  NuRD homologues in embryonic protein extracts  Nuclear protein extracts were prepared from (A) 2–4 h and (B) 0–12 h old embryos, respectively and size-fractionated by FPLC using a Superose 6 column. Proteins from selected fractions were then separated by SDS gel electrophoresis and analysed by Western blotting. (A) During early embryogenesis the long isoform of MBD2/3 (MBD2/3li) cofractionated with MI-2 and MTA-like in fractions 19 to 21 (indicated in red), which correspond to a molecular weight of approx. 1 MDa. The small isoform of MBD2/3 is not expressed at this stage of embryogenesis. (B) During later stages of embryogenesis both isoforms of MBD2/3 cofractionated with MI-2 and MTA-like in fractions 15 to 19 (indicated in red), which correspond to a molecular weight of approx. 2 MDa. The size of marker proteins is shown left and on top, IN indicates input protein.
MBD2/3 is associated with  Drosophila  NuRD proteins
DrosophilaAn association of  Drosophila  MBD2/3 with the NuRD complex has been suggested previously [ 14 , 12 ]. However, these experiments were done in vitro or with protein extracts from tissue culture cells that express only the small isoform of MBD2/3. In addition, only a limited number of NuRD proteins were analysed. To confirm the association between MBD2/3 and NuRD in  Drosophila  embryos we size-fractionated nuclear protein extracts from 2–4 h or 0–12 h wild type embryos, respectively, by Superose 6 gel filtration chromatography. Proteins from individual fractions were then separated by standard SDS-PAGE and analysed by Western blotting using antibodies directed against known homologous subunits of the NuRD complex. MI-2 and MTA-like, which are homologues of subunits that are specific for vertebrate NuRD complexes [ 24 , 3 ] cofractionated with an apparent molecular weight of 1 MDa when extracts derived from early embryos (2–4 h) were applied (Fig.  1A , fractions 19 to 21). When extracts of late embryos (0–12 h) were analysed, MI-2 and MTA-like were detected in fractions 15 to 19, which correspond to an apparent molecular weight of approx. 2 MDa (Fig.  1B ). This result indicates that the formation of NuRD is developmentally regulated during embryogenesis. Additionally, we found a precise cofractionation of the large isoform of MBD2/3 (Fig.  1 , MBD2/3li) with MI-2 and MTA-like in both cases. The small MBD2/3 isoform (Fig.  1B , MBD2/3si) present in extracts derived from late embryos likewise cofractionated with MI-2 and MTA-like. This suggests a close association of both MBD2/3 isoforms with the  Drosophila  NuRD complex. The RPD3 and p55 proteins also cofractionated with MBD2/3 isoforms, but they were also found in fractions of lower molecular weight (Fig.  1 ). This is in agreement with the fact that both RPD3 and p55 are also components of other chromatin related complexes [ 25 , 26 ].DrosophilaDrosophilaDrosophilaInteractions between MBD2/3 and NuRD homologues To analyse the association between MBD2/3 and the NuRD complex in more detail and to identify direct NuRD interaction partner(s) of MBD2/3 we first used a GST-pulldown assay. In order to eliminate unspecific interactions with NuRD proteins, we established highly stringent assay conditions. This was achieved by assessing the interactions between radioactively labeled SV40 large T antigen (Fig.  2A , large T) and a number of control GST fusion proteins. When we used an incubation and washing buffer with high salt and detergent concentrations, significant amounts of SV40 large T were precipitated by p53 only (Fig.  2A ), which is known to be a strong interactor of the large T antigen [ 27 ]. Residual binding detected with some of the other proteins (Fig.  2A ) was considered background. Figure 2 Interactions between MBD2/3 and NuRD homologues in a GST-pulldown assay  (A) Analysis of interactions between radioactively labelled SV40 large T antigen (large T) and a number of control GST fusion proteins under stringent buffer conditions. Significant amounts of SV40 large T were only precipitated by p53. The faint bands seen with some of the other proteins were considered background. (B) GST-MBD2/3 fusion proteins for long and small isoforms (MBD2/3li, MBD2/3si, respectively) efficiently precipitated radioactively labelled MBD2/3 long isoform and p55 (solid arrowhead). Similarly, MI-2 protein could be precipitated by the small GST-MBD2/3 isoform (solid arrowhead). No interaction could be observed between long MBD2/3 or small MBD2/3 isoforms and MTA-like or RPD3 (open arrowheads). (C) A MBD2/3-specific antibody immunoprecipitates p55, but not GAGA factor from embryonic nuclear extracts. No proteins were detectable in control immunoprecipitations with a myc-specific antibody. (D) Size-fractionation of baculovirus-expressed MBD2/3 long isoform and RPD3. Baculovirus-expressed MBD2/3li elutes in fractions that significantly exceed the calculated molecular weight of the protein (36 kDa), thus indicating that MBD2/3li efficiently multimerizes in solution. This effect appeared to be specific for MBD2/3 and was not observed with baculovirus-expressed RPD3 protein (58kDa). The size of marker proteins is shown left and on top, IN indicates input protein. We then performed the MBD2/3-NuRD interaction assay using these conditions and observed strong interactions only between the long and small MBD2/3 isoforms (Fig.  2B , MBD2/3li and MBD2/3si, respectively) and p55. To confirm the association between MBD2/3 and p55 in  Drosophila  embryos we immunoprecipitated nuclear extracts with MBD2/3-specific antibodies and with myc-specific control antibodies. Precipitates were analysed by Western blot for the presence of p55 and GAGA factor. This revealed a specific association of MBD2/3 with the  Drosophila  NuRD homologue p55, but not with the unrelated GAGA factor (Fig.  2C ). A weaker interaction could also be detected between the small MBD2/3 isoform and MI-2 (Fig.  2B ). Neither RPD3 nor MTA-like binding exceeded the background level defined by our pilot experiment with SV40 large T protein (Fig.  2B ). Our results thus identified p55 and MI-2 as the direct interaction partners of MBD2/3 in the NuRD complex. In addition, our results from the GST-pulldown assay indicate that  Drosophila  MBD2/3 forms dimers or multimers, similar to mammalian MBD2 and MBD3 [ 28 ]. To discriminate between the latter two possibilities we expressed a recombinant long MBD2/3 isoform and RPD3 in insect cells using a baculovirus expression system. Extracts from infected cells were subjected to Superdex 200 gel filtration and fractions were analysed by Western blot. The 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 exists as monomers or dimers in solution (Fig.  2D ). In contrast, the 36 kDa MBD2/3 isoform showed a strikingly different elution profile (Fig.  2D ). The long MBD2/3 isoform eluted in a broad peak ranging from more than 158 kDa up to high molecular weight fractions (>400 kDa). No MBD2/3 was detected in fractions corresponding to the expected size of MBD2/3 monomers or dimers. This result is consistent with our earlier observation that MBD2/3 forms distinct aggregates in embryonic nuclei [ 15 ] and suggests that the protein efficiently oligomerises to form high-molecular weight complexes in solution. To confirm these interactions in an independent set of experiments we also analysed the association between MBD2/3 isoforms and the NuRD proteins in a yeast two-hybrid assay. In agreement with our previous results, growth on highly selective (Fig.  3A , panel H) plates was only observed upon co-expression of the MBD2/3 small isoform and either MI-2, MBD2/3 long isoform or p55, and upon co-expression of both MBD2/3 isoforms. These interactions were confirmed by X-Gal staining of filter-lifted yeast colonies (Fig.  3A , panel X). In these assays the two MBD2/3 isoforms differed only with regard to MI-2 binding (Fig.  3A ). However, differential binding could be eliminated by a slight reduction in the stringency of the assay (Fig.  3A , panel M), which suggested that both isoforms of MBD2/3 are capable of interacting with MI-2. Consistent with our GST-pulldown assays no interaction could be detected between the MBD2/3 long isoform and RPD3 or MTA-like (Fig.  3A ). Figure 3 Interactions between MBD2/3 and NuRD homologues in a yeast two-hybrid assay  (A) An MBD2/3 long isoform bait efficiently transactivated reporter gene transcription in yeast expressing p55 or MBD2/3 small isoform prey constructs, respectively. A weaker transactivation could be seen upon expression of a MI-2 prey construct (panel M). An MBD2/3 small isoform bait transactivated reporter gene transcription co-expressing a MI-2 prey also under high-stringent conditions (panel H). No transactivation could be observed with MTA-like or RPD3 preys. N shows growth on non-selective plates, M on medium-stringency plates and H on high-stringency plates. X indicates X-gal staining of colonies grown on non-selective plates. (B and C) Delineation of interaction domains in a yeast two-hybrid assay. The methyl-DNA binding domain (MBD) is highlighted in red, the  Drosophila  specific domain (DSD) in green and the C-terminal coiled-coil domain (cc) in yellow. (B) The C-terminal region of MBD2/3 interacts with p55. The full-length p55 construct was used as a bait and various deletion mutants of MBD2/3 were used as preys. This identified the region between amino acids 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain. (C) Deletion of the coiled-coil domain abolishes interactions between MBD2/3 small isoform and MI-2 under high stringent conditions. Next we sought to delineate the domains that mediate the association between the MBD2/3 isoforms and their interacting proteins. To this end, we generated several MBD2/3 deletion mutants and tested their interaction with other proteins in a yeast two-hybrid assay. In a first series of experiments we tested the MBD2/3 mutants for their ability to interact with p55. This identified the region between residues 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain (Fig.  3B ). We also delineated the domain that mediates the interaction with MI-2. Our experiments revealed a strongly reduced interaction between a MBD2/3 small isoform derivative lacking the coiled-coil domain and MI-2 (Fig.  3C ). It has been shown previously that vertebrate MBD2 and MBD3 form homo- and heterodimers via their N-terminal MBD domain and their C-terminal coiled-coil like sequences [ 28 ]. Our findings might reflect a direct interaction between the coiled-coil domain and MI-2 or a requirement for efficient MBD2/3 multimerization for MI-2 interaction. In conclusion, our results identify distinct regions in the MBD2/3 protein that mediate protein-protein interactions with other NuRD proteins. In order to analyse the architecture of the NuRD complex, we performed a detailed yeast two-hybrid analysis including additional bait and prey constructs for NuRD proteins. Bait constructs for MI-2, RPD3, p55 and both MBD2/3 isoforms and prey constructs for MTA-like, RPD3, p55, and both MBD2/3 isoforms were co-transformed in all possible combinations and transformation was confirmed by growth on non-selective plates (Fig.  4A ). Colonies were then replicated onto highly selective plates that allow growth only upon interaction between the two expressed proteins (Fig.  4B ). Finally, we performed a filter lift assay followed by X-Gal staining to confirm the interactions (Fig.  4C ). In addition to the results presented above, we found strong interactions between p55 and all NuRD proteins. We also observed a homotypic interaction for p55 but no homotypic interaction for RPD3. To confirm the specificity of the interactions, we tested most of the constructs reciprocally by changing the bait and prey vectors for expression of NuRD proteins and MBD2/3 isoforms. This data allows the establishment of a more detailed model of the NuRD complex. Fig.  4D  summarizes the data from all experiments. p55 interacted with MI-2, MTA-like, RPD3, and both MBD2/3 isoforms. The small isoform of MBD2/3 interacted with MI-2 as well as with the large MBD2/3 isoform. Homodimerization of p55 and MBD2/3 is not shown in the model. Figure 4 Analysis of the architecure of NuRD  (A) Yeast colonies growing on non-selective plates carrying bait (left panel) and prey (top panel) NuRD and MBD2/3 constructs. (B) Replica plate with high-selective medium. Only yeast colonies with constructs encoding interacting proteins are able to grow. MBD2/3li Δ59-339  represents a truncated MBD2/3li construct that was used to confirm the specificity of the interaction between p55 and MBD2/3. (C) X-Gal staining of yeast colonies to confirm interactions. (D) Schematic illustration of protein-protein interactions in the NuRD complex. Homodimerization of p55 and MBD2/3 are not shown.
Interactions between MBD2/3 and NuRD homologues
To analyse the association between MBD2/3 and the NuRD complex in more detail and to identify direct NuRD interaction partner(s) of MBD2/3 we first used a GST-pulldown assay. In order to eliminate unspecific interactions with NuRD proteins, we established highly stringent assay conditions. This was achieved by assessing the interactions between radioactively labeled SV40 large T antigen (Fig.  2A , large T) and a number of control GST fusion proteins. When we used an incubation and washing buffer with high salt and detergent concentrations, significant amounts of SV40 large T were precipitated by p53 only (Fig.  2A ), which is known to be a strong interactor of the large T antigen [ 27 ]. Residual binding detected with some of the other proteins (Fig.  2A ) was considered background.We then performed the MBD2/3-NuRD interaction assay using these conditions and observed strong interactions only between the long and small MBD2/3 isoforms (Fig.  2B , MBD2/3li and MBD2/3si, respectively) and p55. To confirm the association between MBD2/3 and p55 in  Drosophila  embryos we immunoprecipitated nuclear extracts with MBD2/3-specific antibodies and with myc-specific control antibodies. Precipitates were analysed by Western blot for the presence of p55 and GAGA factor. This revealed a specific association of MBD2/3 with the  Drosophila  NuRD homologue p55, but not with the unrelated GAGA factor (Fig.  2C ).DrosophilaDrosophilaA weaker interaction could also be detected between the small MBD2/3 isoform and MI-2 (Fig.  2B ). Neither RPD3 nor MTA-like binding exceeded the background level defined by our pilot experiment with SV40 large T protein (Fig.  2B ). Our results thus identified p55 and MI-2 as the direct interaction partners of MBD2/3 in the NuRD complex. In addition, our results from the GST-pulldown assay indicate that  Drosophila  MBD2/3 forms dimers or multimers, similar to mammalian MBD2 and MBD3 [ 28 ]. To discriminate between the latter two possibilities we expressed a recombinant long MBD2/3 isoform and RPD3 in insect cells using a baculovirus expression system. Extracts from infected cells were subjected to Superdex 200 gel filtration and fractions were analysed by Western blot. The 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 exists as monomers or dimers in solution (Fig.  2D ). In contrast, the 36 kDa MBD2/3 isoform showed a strikingly different elution profile (Fig.  2D ). The long MBD2/3 isoform eluted in a broad peak ranging from more than 158 kDa up to high molecular weight fractions (>400 kDa). No MBD2/3 was detected in fractions corresponding to the expected size of MBD2/3 monomers or dimers. This result is consistent with our earlier observation that MBD2/3 forms distinct aggregates in embryonic nuclei [ 15 ] and suggests that the protein efficiently oligomerises to form high-molecular weight complexes in solution.DrosophilaTo confirm these interactions in an independent set of experiments we also analysed the association between MBD2/3 isoforms and the NuRD proteins in a yeast two-hybrid assay. In agreement with our previous results, growth on highly selective (Fig.  3A , panel H) plates was only observed upon co-expression of the MBD2/3 small isoform and either MI-2, MBD2/3 long isoform or p55, and upon co-expression of both MBD2/3 isoforms. These interactions were confirmed by X-Gal staining of filter-lifted yeast colonies (Fig.  3A , panel X). In these assays the two MBD2/3 isoforms differed only with regard to MI-2 binding (Fig.  3A ). However, differential binding could be eliminated by a slight reduction in the stringency of the assay (Fig.  3A , panel M), which suggested that both isoforms of MBD2/3 are capable of interacting with MI-2. Consistent with our GST-pulldown assays no interaction could be detected between the MBD2/3 long isoform and RPD3 or MTA-like (Fig.  3A ).Next we sought to delineate the domains that mediate the association between the MBD2/3 isoforms and their interacting proteins. To this end, we generated several MBD2/3 deletion mutants and tested their interaction with other proteins in a yeast two-hybrid assay. In a first series of experiments we tested the MBD2/3 mutants for their ability to interact with p55. This identified the region between residues 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain (Fig.  3B ). We also delineated the domain that mediates the interaction with MI-2. Our experiments revealed a strongly reduced interaction between a MBD2/3 small isoform derivative lacking the coiled-coil domain and MI-2 (Fig.  3C ). It has been shown previously that vertebrate MBD2 and MBD3 form homo- and heterodimers via their N-terminal MBD domain and their C-terminal coiled-coil like sequences [ 28 ]. Our findings might reflect a direct interaction between the coiled-coil domain and MI-2 or a requirement for efficient MBD2/3 multimerization for MI-2 interaction. In conclusion, our results identify distinct regions in the MBD2/3 protein that mediate protein-protein interactions with other NuRD proteins.In order to analyse the architecture of the NuRD complex, we performed a detailed yeast two-hybrid analysis including additional bait and prey constructs for NuRD proteins. Bait constructs for MI-2, RPD3, p55 and both MBD2/3 isoforms and prey constructs for MTA-like, RPD3, p55, and both MBD2/3 isoforms were co-transformed in all possible combinations and transformation was confirmed by growth on non-selective plates (Fig.  4A ). Colonies were then replicated onto highly selective plates that allow growth only upon interaction between the two expressed proteins (Fig.  4B ). Finally, we performed a filter lift assay followed by X-Gal staining to confirm the interactions (Fig.  4C ). In addition to the results presented above, we found strong interactions between p55 and all NuRD proteins. We also observed a homotypic interaction for p55 but no homotypic interaction for RPD3. To confirm the specificity of the interactions, we tested most of the constructs reciprocally by changing the bait and prey vectors for expression of NuRD proteins and MBD2/3 isoforms. This data allows the establishment of a more detailed model of the NuRD complex. Fig.  4D  summarizes the data from all experiments. p55 interacted with MI-2, MTA-like, RPD3, and both MBD2/3 isoforms. The small isoform of MBD2/3 interacted with MI-2 as well as with the large MBD2/3 isoform. Homodimerization of p55 and MBD2/3 is not shown in the model.Discussion It has been previously suggested that MBD2/3 is associated with the  Drosophila  NuRD complex [ 14 ]. This study determined that the small isoform MBD2/3 coelutes with some putative  Drosophila  NuRD subunits during fractionation of extracts derived from a  Drosophila  cell line. We have now extended this analysis to show that both isoforms of MBD2/3 coelute with NuRD homologues during fractionation of embryonic extracts. This data provides further evidence for a direct interaction between MBD2/3 and the NuRD complex. Using several independent assays, we have demonstrated that MBD2/3 engages in homotypic interactions to form multimers. This effect is consistent with the formation of foci in embryonic nuclei [ 15 ] and also reminiscent of the interactions described for vertebrate MBD2 and MBD3 [ 28 ]. In addition, our data provides new insights into the association between MBD2/3 and NuRD. For example, we have shown that p55 appears to be the primary interaction partner of MBD2/3. We also observed a strong interaction between the small isoform MBD2/3 and MI-2, but not between MBD2/3 small isoform and MTA-like or RPD3. Additionally, we found interactions between p55 and all NuRD proteins, as well as a p55 homotypic interaction. The last finding is consistent with the fact that in the vertebrate NuRD complex the two p55 homologues RbAp46 and RbAp48 were identified as integral components [ 3 ]. We note that the vertebrate NuRD complex also contains the two RPD3 homologues HDAC1 and HDAC2 [ 3 ]; unexpectedly, we were not able to detect any homotypic interaction of RPD3 in the yeast two-hybrid assay. One explanation could be that the dose of the two co-expressed RPD3 bait and prey proteins could interfere with the reporter gene activity due to their inherent histone deacetylation activity. However, the gel filtration assay revealed that the 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 can interact homotypically (Fig.  3C ). It is possible that more complex interactions are involved in the assembly of the NuRD complex but they might not have been detectable under the stringent conditions of our assays. The interaction between MBD2/3 and MI-2 detected in our assays could also contribute to the specific association between MBD2/3 and the NuRD complex. The interaction between MBD2/3 and p55 could promote the assembly of specialized chromatin structures in the fly. p55 is a WD-40 repeat protein that is involved in many aspects of chromatin organization [ 29 ]. For example, p55 has been shown to be a component of the  Drosophila  CAF-1 complex that promotes nucleosome assembly [ 22 ]. In addition, p55 is also contained in the NURF chromatin remodelling complex [ 30 ] and in the E(Z) complex that regulates homeotic gene expression [ 25 , 26 ]. A mutant  Drosophila  allele for  p55  is not available, but results obtained from  Arabidopsis  mutants with decreased levels of a p55 homologue indicate that the protein plays an important role in stabilizing epigenetic chromatin structures [ 31 ].
Discussion
It has been previously suggested that MBD2/3 is associated with the  Drosophila  NuRD complex [ 14 ]. This study determined that the small isoform MBD2/3 coelutes with some putative  Drosophila  NuRD subunits during fractionation of extracts derived from a  Drosophila  cell line. We have now extended this analysis to show that both isoforms of MBD2/3 coelute with NuRD homologues during fractionation of embryonic extracts. This data provides further evidence for a direct interaction between MBD2/3 and the NuRD complex.DrosophilaDrosophilaDrosophilaUsing several independent assays, we have demonstrated that MBD2/3 engages in homotypic interactions to form multimers. This effect is consistent with the formation of foci in embryonic nuclei [ 15 ] and also reminiscent of the interactions described for vertebrate MBD2 and MBD3 [ 28 ].In addition, our data provides new insights into the association between MBD2/3 and NuRD. For example, we have shown that p55 appears to be the primary interaction partner of MBD2/3. We also observed a strong interaction between the small isoform MBD2/3 and MI-2, but not between MBD2/3 small isoform and MTA-like or RPD3. Additionally, we found interactions between p55 and all NuRD proteins, as well as a p55 homotypic interaction. The last finding is consistent with the fact that in the vertebrate NuRD complex the two p55 homologues RbAp46 and RbAp48 were identified as integral components [ 3 ]. We note that the vertebrate NuRD complex also contains the two RPD3 homologues HDAC1 and HDAC2 [ 3 ]; unexpectedly, we were not able to detect any homotypic interaction of RPD3 in the yeast two-hybrid assay. One explanation could be that the dose of the two co-expressed RPD3 bait and prey proteins could interfere with the reporter gene activity due to their inherent histone deacetylation activity. However, the gel filtration assay revealed that the 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 can interact homotypically (Fig.  3C ).It is possible that more complex interactions are involved in the assembly of the NuRD complex but they might not have been detectable under the stringent conditions of our assays. The interaction between MBD2/3 and MI-2 detected in our assays could also contribute to the specific association between MBD2/3 and the NuRD complex.The interaction between MBD2/3 and p55 could promote the assembly of specialized chromatin structures in the fly. p55 is a WD-40 repeat protein that is involved in many aspects of chromatin organization [ 29 ]. For example, p55 has been shown to be a component of the  Drosophila  CAF-1 complex that promotes nucleosome assembly [ 22 ]. In addition, p55 is also contained in the NURF chromatin remodelling complex [ 30 ] and in the E(Z) complex that regulates homeotic gene expression [ 25 , 26 ]. A mutant  Drosophila  allele for  p55  is not available, but results obtained from  Arabidopsis  mutants with decreased levels of a p55 homologue indicate that the protein plays an important role in stabilizing epigenetic chromatin structures [ 31 ].DrosophilaDrosophilap55ArabidopsisConclusions The goal of this study was to identify the interacting partners of the  Drosophila  methyl-DNA binding protein MBD2/3 within the  Drosophila  NuRD complex. We identified p55 and MI-2 as the primary interacting partners. We also found homo- and heterotypic interactions of the MBD2/3 isoforms, similar to vertebrate MBD2 and MBD3. Additionally, yeast two-hybrid assays revealed that p55 is able to specifically interact with all other NuRD proteins and can form homotypic interactions. Our data provides for the first time information about the architecture of the  Drosophila  NuRD complex. This allows us to develop a structural model of the NuRD complex.
Conclusions
The goal of this study was to identify the interacting partners of the  Drosophila  methyl-DNA binding protein MBD2/3 within the  Drosophila  NuRD complex. We identified p55 and MI-2 as the primary interacting partners. We also found homo- and heterotypic interactions of the MBD2/3 isoforms, similar to vertebrate MBD2 and MBD3. Additionally, yeast two-hybrid assays revealed that p55 is able to specifically interact with all other NuRD proteins and can form homotypic interactions. Our data provides for the first time information about the architecture of the  Drosophila  NuRD complex. This allows us to develop a structural model of the NuRD complex.DrosophilaDrosophilaDrosophilaMethods DNA constructs Constructs for the yeast two-hybrid assay were generated by PCR amplification from cDNA clones [ 32 ] using specific primers (Tab. 1) with an attached restriction endonuclease target site for cloning. The PCR products were subsequently cloned into pGBKT7 or pGADT7 (Clontech) using standard procedures. All constructs were sequenced and in vitro translated to confirm the expression of corresponding proteins. Additionally, the bait constructs were expressed in yeast and expression of the fusion proteins was confirmed by Western analysis. Antibodies The following antibodies have been described previously: rabbit anti-MI-2 [ 20 ], rabbit anti-MTA1 [ 19 , 4 ], rabbit anti-RPD3 [ 20 ], rabbit anti-p55 [ 22 ], rabbit anti-MBD2/3 [ 14 ] and rabbit anti-GAGA [ 33 ]. Immunoprecipitations Immunoprecipitations were caried out in 300 mM KCl supplemented with 0.2 % NP-40. Rabbit anti-MBD2/3 or mouse anti-myc (Clontech) antibodies were added to 75 μl of nuclear extracts prepared as mentioned below and incubated for 12 h at 4°C. Protein G beads (Amersham) were blocked in 3 mg/ml BSA for 20 min at room temperature, washed three times with 300 mM KCl, 0.2 % NP-40 and added to the samples. Incubation was carried out for an additional 1 h at 4°C. The beads were collected by centrifugation and washed four times in 1 ml 300 mM KCl, 0.2 % NP-40. The beads were resuspended in loading buffer for SDS-PAGE and vigorously vortexed for 15 sec. The immunoprecipitates were separated by SDS-PAGE, without futher boiling, transferred to a PVDF membrane and probed with antibodies against RPD3, p55 and GAGA factor, respectively. Preparation of protein extracts and gel filtration For baculovirus expression, the MBD2/3 cDNA [ 32 ] was subcloned into the pVL1392 baculovirus transfer vector. Transfer vector and linearised baculovirus DNA were cotransfected into Sf9 cells using the Bac'n'Blue transfection kit (Invitrogen) and recombinant virus was amplified according to the manufacturer's instructions. Whole cell extracts of infected Sf9 cells were generated by resuspending cell pellets in lysis buffer (20 mM Hepes pH 7.6, 200 mM KCl, 0.1 % NP40), incubation on ice for 10 min, three freeze/thaw cycles and sonication. Nuclear extracts from  Drosophila  embryos were prepared as described previously [ 20 ]. Extracts were cleared by centrifugation and passaged through a 0.2 μm filter. 200 μl of cleared extract was applied to Superdex 200 HR 10/30 or Superose 6 HR 10/30 gel filtration columns (Amersham Pharmacia) and resolved in 20 mM Hepes pH 7.6 and 300 mM KCl on an Äkta Purifier system (Amersham Pharmacia) according to the manufacturer's instructions. GST-pulldown assays 35 S-methionine-labelled proteins were generated by in vitro trancription/translation of pGADT7_T, pGADT7_Mi-2, pGADT7_MTA-like, pGADT7_Rpd3, pGADT7_p55, pGADT7_MBD2/3 long isoform and pGADT7_MBD2/3 small isoform using the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. GST-MBD2/3 long isoform and GST-MBD2/3 small isoform fusionproteins were obtained by cloning the coding region of the two isoforms in pGEX4T1 (Amersham) and subsequent expressing of the constructs in BL-21 bacteria according to the manufacturer's protocol. GST-MBD2a was obtained from Hidetoshi Fujita [ 34 ]. GST-pull downs were performed under the following conditions: As incubation and washing buffer we used 20 mM HEPES, pH 7.8, 300 mM NaCl, 0.1 % desoxycholate, 0.1 % IGEPAL, 10 % glycerol. The radioactively labelled proteins were incubated in incubation buffer for 3 h at room temperature with GST fusion proteins coupled to Sepharose 4B (Amersham) or with GST alone coupled to Sepharose 4B. Beads were then washed five times for 10 min at room temperature. After the last washing step, beads were boiled in SDS loading buffer for 10 min and loaded onto standard SDS-polyacrylamide gels. After separation by SDS-PAGE, proteins were blotted onto a PVDF membrane, which was stained with Ponceau S, dried and exposed on X-ray films. Yeast two-hybrid assays The Matchmaker Two-Hybrid system 3 (Clontech) was used for all experiments, according to the manufacturer's instructions. The AH109 strain was used as host and transformed with various constructs (see above) using standard procedures. Transformants were selected on SD/-Ade/-His/-Leu/-Trp plates.
Methods
DNA constructs Constructs for the yeast two-hybrid assay were generated by PCR amplification from cDNA clones [ 32 ] using specific primers (Tab. 1) with an attached restriction endonuclease target site for cloning. The PCR products were subsequently cloned into pGBKT7 or pGADT7 (Clontech) using standard procedures. All constructs were sequenced and in vitro translated to confirm the expression of corresponding proteins. Additionally, the bait constructs were expressed in yeast and expression of the fusion proteins was confirmed by Western analysis.
DNA constructs
Constructs for the yeast two-hybrid assay were generated by PCR amplification from cDNA clones [ 32 ] using specific primers (Tab. 1) with an attached restriction endonuclease target site for cloning. The PCR products were subsequently cloned into pGBKT7 or pGADT7 (Clontech) using standard procedures. All constructs were sequenced and in vitro translated to confirm the expression of corresponding proteins. Additionally, the bait constructs were expressed in yeast and expression of the fusion proteins was confirmed by Western analysis.Antibodies The following antibodies have been described previously: rabbit anti-MI-2 [ 20 ], rabbit anti-MTA1 [ 19 , 4 ], rabbit anti-RPD3 [ 20 ], rabbit anti-p55 [ 22 ], rabbit anti-MBD2/3 [ 14 ] and rabbit anti-GAGA [ 33 ].
Antibodies
The following antibodies have been described previously: rabbit anti-MI-2 [ 20 ], rabbit anti-MTA1 [ 19 , 4 ], rabbit anti-RPD3 [ 20 ], rabbit anti-p55 [ 22 ], rabbit anti-MBD2/3 [ 14 ] and rabbit anti-GAGA [ 33 ].Immunoprecipitations Immunoprecipitations were caried out in 300 mM KCl supplemented with 0.2 % NP-40. Rabbit anti-MBD2/3 or mouse anti-myc (Clontech) antibodies were added to 75 μl of nuclear extracts prepared as mentioned below and incubated for 12 h at 4°C. Protein G beads (Amersham) were blocked in 3 mg/ml BSA for 20 min at room temperature, washed three times with 300 mM KCl, 0.2 % NP-40 and added to the samples. Incubation was carried out for an additional 1 h at 4°C. The beads were collected by centrifugation and washed four times in 1 ml 300 mM KCl, 0.2 % NP-40. The beads were resuspended in loading buffer for SDS-PAGE and vigorously vortexed for 15 sec. The immunoprecipitates were separated by SDS-PAGE, without futher boiling, transferred to a PVDF membrane and probed with antibodies against RPD3, p55 and GAGA factor, respectively.
Immunoprecipitations
Immunoprecipitations were caried out in 300 mM KCl supplemented with 0.2 % NP-40. Rabbit anti-MBD2/3 or mouse anti-myc (Clontech) antibodies were added to 75 μl of nuclear extracts prepared as mentioned below and incubated for 12 h at 4°C. Protein G beads (Amersham) were blocked in 3 mg/ml BSA for 20 min at room temperature, washed three times with 300 mM KCl, 0.2 % NP-40 and added to the samples. Incubation was carried out for an additional 1 h at 4°C. The beads were collected by centrifugation and washed four times in 1 ml 300 mM KCl, 0.2 % NP-40. The beads were resuspended in loading buffer for SDS-PAGE and vigorously vortexed for 15 sec. The immunoprecipitates were separated by SDS-PAGE, without futher boiling, transferred to a PVDF membrane and probed with antibodies against RPD3, p55 and GAGA factor, respectively.Preparation of protein extracts and gel filtration For baculovirus expression, the MBD2/3 cDNA [ 32 ] was subcloned into the pVL1392 baculovirus transfer vector. Transfer vector and linearised baculovirus DNA were cotransfected into Sf9 cells using the Bac'n'Blue transfection kit (Invitrogen) and recombinant virus was amplified according to the manufacturer's instructions. Whole cell extracts of infected Sf9 cells were generated by resuspending cell pellets in lysis buffer (20 mM Hepes pH 7.6, 200 mM KCl, 0.1 % NP40), incubation on ice for 10 min, three freeze/thaw cycles and sonication. Nuclear extracts from  Drosophila  embryos were prepared as described previously [ 20 ]. Extracts were cleared by centrifugation and passaged through a 0.2 μm filter. 200 μl of cleared extract was applied to Superdex 200 HR 10/30 or Superose 6 HR 10/30 gel filtration columns (Amersham Pharmacia) and resolved in 20 mM Hepes pH 7.6 and 300 mM KCl on an Äkta Purifier system (Amersham Pharmacia) according to the manufacturer's instructions.
Preparation of protein extracts and gel filtration
For baculovirus expression, the MBD2/3 cDNA [ 32 ] was subcloned into the pVL1392 baculovirus transfer vector. Transfer vector and linearised baculovirus DNA were cotransfected into Sf9 cells using the Bac'n'Blue transfection kit (Invitrogen) and recombinant virus was amplified according to the manufacturer's instructions. Whole cell extracts of infected Sf9 cells were generated by resuspending cell pellets in lysis buffer (20 mM Hepes pH 7.6, 200 mM KCl, 0.1 % NP40), incubation on ice for 10 min, three freeze/thaw cycles and sonication. Nuclear extracts from  Drosophila  embryos were prepared as described previously [ 20 ]. Extracts were cleared by centrifugation and passaged through a 0.2 μm filter. 200 μl of cleared extract was applied to Superdex 200 HR 10/30 or Superose 6 HR 10/30 gel filtration columns (Amersham Pharmacia) and resolved in 20 mM Hepes pH 7.6 and 300 mM KCl on an Äkta Purifier system (Amersham Pharmacia) according to the manufacturer's instructions.DrosophilaGST-pulldown assays 35 S-methionine-labelled proteins were generated by in vitro trancription/translation of pGADT7_T, pGADT7_Mi-2, pGADT7_MTA-like, pGADT7_Rpd3, pGADT7_p55, pGADT7_MBD2/3 long isoform and pGADT7_MBD2/3 small isoform using the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. GST-MBD2/3 long isoform and GST-MBD2/3 small isoform fusionproteins were obtained by cloning the coding region of the two isoforms in pGEX4T1 (Amersham) and subsequent expressing of the constructs in BL-21 bacteria according to the manufacturer's protocol. GST-MBD2a was obtained from Hidetoshi Fujita [ 34 ]. GST-pull downs were performed under the following conditions: As incubation and washing buffer we used 20 mM HEPES, pH 7.8, 300 mM NaCl, 0.1 % desoxycholate, 0.1 % IGEPAL, 10 % glycerol. The radioactively labelled proteins were incubated in incubation buffer for 3 h at room temperature with GST fusion proteins coupled to Sepharose 4B (Amersham) or with GST alone coupled to Sepharose 4B. Beads were then washed five times for 10 min at room temperature. After the last washing step, beads were boiled in SDS loading buffer for 10 min and loaded onto standard SDS-polyacrylamide gels. After separation by SDS-PAGE, proteins were blotted onto a PVDF membrane, which was stained with Ponceau S, dried and exposed on X-ray films.
GST-pulldown assays
35 S-methionine-labelled proteins were generated by in vitro trancription/translation of pGADT7_T, pGADT7_Mi-2, pGADT7_MTA-like, pGADT7_Rpd3, pGADT7_p55, pGADT7_MBD2/3 long isoform and pGADT7_MBD2/3 small isoform using the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. GST-MBD2/3 long isoform and GST-MBD2/3 small isoform fusionproteins were obtained by cloning the coding region of the two isoforms in pGEX4T1 (Amersham) and subsequent expressing of the constructs in BL-21 bacteria according to the manufacturer's protocol. GST-MBD2a was obtained from Hidetoshi Fujita [ 34 ]. GST-pull downs were performed under the following conditions: As incubation and washing buffer we used 20 mM HEPES, pH 7.8, 300 mM NaCl, 0.1 % desoxycholate, 0.1 % IGEPAL, 10 % glycerol. The radioactively labelled proteins were incubated in incubation buffer for 3 h at room temperature with GST fusion proteins coupled to Sepharose 4B (Amersham) or with GST alone coupled to Sepharose 4B. Beads were then washed five times for 10 min at room temperature. After the last washing step, beads were boiled in SDS loading buffer for 10 min and loaded onto standard SDS-polyacrylamide gels. After separation by SDS-PAGE, proteins were blotted onto a PVDF membrane, which was stained with Ponceau S, dried and exposed on X-ray films.Yeast two-hybrid assays The Matchmaker Two-Hybrid system 3 (Clontech) was used for all experiments, according to the manufacturer's instructions. The AH109 strain was used as host and transformed with various constructs (see above) using standard procedures. Transformants were selected on SD/-Ade/-His/-Leu/-Trp plates.
Yeast two-hybrid assays
The Matchmaker Two-Hybrid system 3 (Clontech) was used for all experiments, according to the manufacturer's instructions. The AH109 strain was used as host and transformed with various constructs (see above) using standard procedures. Transformants were selected on SD/-Ade/-His/-Leu/-Trp plates.Authors' contributions KK and JM carried out the molecular interaction studies. AB carried out the gel filtration experiments. JM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Table 1 PCR primers used for the cloning of recombinant plasmids MBD-BD-F: gga att cat gca aat gaa ccc gag cgt c MBD-BD-R: tcc ccc ggg tgt ctt gag tgc atc ctg cag Mi2-BD-F: cgc cat atg atg gca tcg gag gaa gag aat gac Mi2-BD-R: gcg gcc tcc atg gcc gac gcc gga att att cga tag c MTA-AD-F: ccg gaa ttc atg gcc aca aat atg tat cga gtc gg MTA-AD-R: tcc ggg ccc ggt gac act ata gaa ctc gag Rpd3-BD-F: atg gcc atg gat gca gtc tca aca gc Rpd3-BD-R: ggc cgc tgc aga atg ttg ttc tcc ttg gcg Rpd3-AD-F: cgc tca tat gat gca gtc tca cag c Rpd3-AD-R: gca gct cga gaa tgt tgt tct cct tgg cg p55-BD-R: ggc tca atc ttt ggt tat ggc gaa ttg gat ccg cg p55-AD-F: ccg gaa ttc atg gtg gat cgc agc g p55-AD-R: ggc gag ctc tta agc ggt att ggt ttc taa ctc gg MBD-AD-F: gga att cat gca aat gaa ccc gag cgt c MBD-AD-R: ccg ctc gag tgt ctt gag tgc atc ctg cag MBD-AD-Δ59-339: ccg ctc gag cca cct tgt tat tgt tgt tgt tgc BD designates primers used for the binding domain-constructs, AD designates primers used for the activation domain-constructs. F indicates forward primers, R indicates reverse primers.
Authors' contributions
KK and JM carried out the molecular interaction studies. AB carried out the gel filtration experiments. JM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.PCR primers used for the cloning of recombinant plasmidsBD designates primers used for the binding domain-constructs, AD designates primers used for the activation domain-constructs. F indicates forward primers, R indicates reverse primers.