Research Reports Identification and mapping of DNA binding proteins target sequences in long genomic regions by two-dimensional EMSA Igor P. Chernov, Sergey B. Akopov, Lev G. Nikolaev, and Eugene D. Sverdlov BioTechniques 41:90-96 (July 2006) doi 10.2144/000112197 Specific binding of nuclear proteins, in particular transcription factors, to target DNA sequences is a major mechanism of genome functioning and gene expression regulation in eukaryotes. Therefore, identification and mapping specific protein target sites (PTS) is necessary for understanding genomic regulation. Here we used a novel two-dimensional electrophoretic mobility shift assay (2D- EMSA) procedure for identification and mapping of 52 PTS within a 563-kb human genome region located between the FXYD5 and TZFP genes. The PTS occurred with approximately equal frequency within unique and repetitive genomic regions. PTS belonging to unique sequences tended to group together within gene introns and close to their 5′ and 3′ ends, whereas PTS located within repeats were evenly distributed between transcribed and intragenic regions. INTRODUCTION mechanism of genome functioning identification of a whole set of specific and regulation in eukaryotes (5), that PTS and grouping them according to The publication of the human makes identification and mapping their functional role and interactions genome sequence (1,2) and sequences of specific protein target sites (PTS) with other regulatory units. The result of other metazoan genomes greatly necessary for understanding genomic should be a protein binding map of facilitated positioning and analysis of regulation. To date, several approaches extended genomic regions or even various genomic functional elements to unbiased mapping of PTS have been whole genomes, ideally a dynamic and first of all coding sequences (3,4). proposed and used. The most widely map depending on cell origin, environ- At the same time, a complete functional used is a chromatin immunopre- mental conditions, and other factors. annotation of sequenced eukaryotic cipitation-on-a-chip (ChIP-on-chip) Recently, we proposed experi- genomes is supposed to include technique that allowed to map target mental approaches for identification positions of all noncoding regulatory sites for the NF-κB (6) and CREB and mapping of nuclear matrix binding elements. Unfortunately, experi- (7) transcription factors across human regions (S/MARs) (14) within a 1-Mb mental data on genomic positions of a chromosome 22 and the Sp1, c-Myc, human chromosome 19 locus between multitude of regulatory sequences, like and p53 factors across human chromo- the FXYD5 and COX7A1 markers enhancers, promoters, transcription somes 21 and 22 (8). Another experi- (15). The locus contains 45 Reference terminators, and replication origins, are mental approach named DamID (9) Sequence (16) genes expressed with very limited, especially at the whole was recently used for mapping GAGA different tissue specificities and therefore genome level. In general, most genomic (10), Myc, Max, and Mad/Mnt target could be a good model for the study regulatory elements (e.g., enhancers) sites across the whole Drosophila of the mammalian genome regulatory are gene-, tissue-, or cell-specific, and genome (11). It should be noted that network. Here we present an approach prediction of these elements by compu- the both techniques are applicable for high-throughput identification and tational methods is difficult and not only to mapping binding sites of well- mapping of a multitude of PTS within always reliable. Therefore, the devel- characterized transcription factors. a given genomic region. Using this opment of high-throughput experi- Computational identification of approach, we mapped 52 sequences mental approaches to identification PTS is, in turn, strongly limited by the capable of specifically binding Jurkat and mapping of genomic functional lack of experimental data necessary cell nuclear proteins within a 563-kb elements is highly desirable. for development of algorithms and long FXYD5-TZFP human chromosome Specific binding of nuclear proteins validation of the results (12,13). A 19 region, a fragment of the FXYD5- to target DNA sequences is a major general approach should include COX7A1 locus mentioned above. Russian Academy of Sciences, Moscow, Russia 90 ı BioTechniques ı www.biotechniques.com Vol. 41 ı No. 1 ı 2006 Research Reports MATERIALS AND METHODS itated as described (21). To increase nuclear protein extracts (26). Recently, the specificity of selection, the above we proposed a two-dimensional variant 2D-EMSA procedure was repeated of EMSA (2D-EMSA) that allowed Basic Protocols twice. Finally, 2 μL polyacrylamide gel us to identify and map binding sites of Growth and transformation of eluate were PCR-amplified (20 cycles the CTCF transcription factor within a Escherichia coli cells, preparation of of 94°C for 20 s, 60°C for 60 s, and 1-Mb human genome region (21). Here ® plasmid DNA, agarose gel electropho- 72°C for 90 s) and cloned in a pGEM - we present a modification of 2D-EMSA resis, electrophoretic mobility shift assay T vector (Promega, Madison, WI, USA) that allows one to obtain and clone DNA (EMSA), and other standard manipula- according to the manufacturer’s recom- fragments capable of binding nuclear tions were performed as described (17). mendations. White colonies (184) were extract proteins of given cells, with the selected and arrayed on a 96-well micro- pattern of the fragments probably being plate. The selected clones were checked � Cells and Nuclear Extract by PCR, and those lacking inserts or producing more than one PCR product A Jurkat cells (acute T cell leukemia, (double inserts) were discarded. TIB-152; ATCC, Manassa, VA, USA) were grown in suspension at 37°C �� One-Dimensional EMSA ���������� and 5% CO2 in RPMI-1640 supple- mented with 10% fetal calf serum, up 6 For EMSA, inserts of individual to approximately 2 × 10 /mL. Nuclear clones were labeled by PCR as extract was isolated as previously described above and purified by described (18) with modifications (19). electrophoresis in a 5% polyacryl- amide gel. EMSA was done essen- Preparation of a Short-Fragment tially as described above with a Library 50,000 counts per minute (cpm) probe, 1 μg nuclear extract protein, and 1 μg DNA of cosmids R30072, R28588, poly(dI-dC)*poly(dI-dC). For compe- F19410, R30879, F24108, F16632, tition experiments, an excess of an R26667, F12426, R28461, F14121, unlabeled probe was added. R31396, F25451, R31076, R28052, ���������������������������������������������� and P1-derived artificial chromosome Sequencing, Computer Analysis, (PAC) PC28130 (kindly provided by A. and Mapping Olsen, Lawrence Livermore National B C Laboratory, Livermore, CA) was Sequencing was done with a ABI isolated and digested with restriction Prism® BigDye™ Terminator v. 3.1 endonucleases Sau3A and Csp6I, kit using an ABI Prism 3100-Avant™ ligated with the library primer 5′-ACT automated sequencer (all from Applied TGAGCTCGAGTATCCATGAACA- Biosystems, Foster City, CA, USA). 3′, and PCR-amplified with the same The sequences obtained were mapped primer as described previously (14,20). by comparison with those deposited in GenBank® using the BLAST (22) Two-Dimensional EMSA server at the National Center for Biotechnology Information (NCBI; For two-dimensional EMSA www.ncbi.nlm.nih.gov/BLAST). The (2D-EMSA), a pool of short DNA data were further analyzed using the Figure 1. The principle and results of two- fragments of a 563-kb FXYD5-TZFP University of California, Santa Cruz dimensional electrophoretic mobility shift region of human chromosome 19 was (UCSC) Human Genome Browser assay (2D-EMSA). (A) General scheme of 2D- EMSA. DNA-protein complexes were initially radioactively labeled by PCR with the (genome.ucsc.edu) (23). separated in a nondenaturing one-dimension library primer and purified as described polyacrylamide gel, and after disruption of the previously (21). The 2D-EMSA was complexes, the DNA fragments released were performed generally as described (21), RESULTS separated in a two-dimension sodium dodecyl sulfate (SDS)-containing gel. The area of spots but instead of purified DNA binding corresponding to target DNA sequences is out- protein, 2.5 μg Jurkat cell nuclear EMSA is one of the most widely lined by the dashed line. (B) The result of 2D- extract protein was added to the initial used methods to explore interactions EMSA with nuclear extract from Jurkat cells EMSA reaction. between DNA and nuclear proteins. The and DNA fragments representing the FXYD5- The gel was then autoradiographed EMSA approach was initially proposed TZFP region of human chromosome 19. (C) Electrophoretic comparison of input DNA frag- overnight, the area containing PTS (see for quantifying interactions between ments with protein target sites (PTS) selected Figure 1A) was excised, cut into small DNA and purified proteins (24,25) and by 2D-EMSA. The most pronounced bands are pieces, and DNA was eluted and precip- was later adapted for crude cellular or marked by arrows. 92 ı BioTechniques ı www.biotechniques.com Vol. 41 ı No. 1 ı 2006 Research Reports characteristic of these cells. Using this outlined by a dashed line (Figure 1A) is Inserts of 120 clones were labeled with approach, we identified and mapped supposed to contain a majority of such 32P, and their ability to specifically bind several tens of potential nuclear protein fragments. nuclear proteins was tested by one- target sequences across an approxi- dimensional EMSA, as described