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

The publication of the human genome sequence (1,2) and sequences of other metazoan genomes greatly facilitated positioning and analysis of various genomic functional elements and first of all coding sequences (3,4). At the same time, a complete functional annotation of sequenced eukaryotic genomes is supposed to include positions of all noncoding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, like enhancers, promoters, transcription terminators, and replication origins, are very limited, especially at the whole genome level. In general, most genomic regulatory elements (e.g., enhancers) are gene-, tissue-, or cell-specific, and prediction of these elements by computational methods is difficult and not always reliable. Therefore, the development of high-throughput experimental approaches to identification and mapping of genomic functional elements is highly desirable.

Specific binding of nuclear proteins to target DNA sequences is a major mechanism of genome functioning and regulation in eukaryotes (5), that makes identification and mapping of specific protein target sites (PTS) necessary for understanding genomic regulation. To date, several approaches to unbiased mapping of PTS have been proposed and used. The most widely used is a chromatin immunopre-cipitation-on-a-chip (ChIP-on-chip) technique that allowed to map target sites for the NF-κB (6) and CREB (7) transcription factors across human chromosome 22 and the Sp1, c-Myc, and p53 factors across human chromosomes 21 and 22 (8). Another experimental approach named DamID (9) was recently used for mapping GAGA (10), Myc, Max, and Mad/Mnt target sites across the whole Drosophila genome (11). It should be noted that the both techniques are applicable only to mapping binding sites of well-characterized transcription factors.

Computational identification of PTS is, in turn, strongly limited by the lack of experimental data necessary for development of algorithms and validation of the results (12,13). A general approach should include identification of a whole set of specific PTS and grouping them according to their functional role and interactions with other regulatory units. The result should be a protein binding map of extended genomic regions or even whole genomes, ideally a dynamic map depending on cell origin, environmental conditions, and other factors.

Recently, we proposed experimental approaches for identification and mapping of nuclear matrix binding regions (S/MARs) (14) within a 1-Mb human chromosome 19 locus between the FXYD5 and COX7A1 markers (15). The locus contains 45 Reference Sequence (16) genes expressed with different tissue specificities and therefore could be a good model for the study of the mammalian genome regulatory network. Here we present an approach for high-throughput identification and mapping of a multitude of PTS within a given genomic region. Using this approach, we mapped 52 sequences capable of specifically binding Jurkat cell nuclear proteins within a 563–kb long FXYD5-TZFP human chromosome 19 region, a fragment of the FXYD5-COX7A1 locus mentioned above.

Materials and Methods

Basic Protocols

Growth and transformation of Escherichia coli cells, preparation of plasmid DNA, agarose gel electropho-resis, electrophoretic mobility shift assay (EMSA), and other standard manipulations were performed as described (17).

Cells and Nuclear Extract

Jurkat cells (acute T cell leukemia, TIB-152; ATCC, Manassa, VA, USA) were grown in suspension at 37°C and 5% CO₂ in RPMI-1640 supplemented with 10% fetal calf serum, up to approximately 2 × 10⁶/mL. Nuclear extract was isolated as previously described (18) with modifications (19).

Preparation of a Short-Fragment Library

DNA of cosmids R30072, R28588, F19410, R30879, F24108, F16632, R26667, F12426, R28461, F14121, R31396, F25451, R31076, R28052, and P1-derived artificial chromosome (PAC) PC28130 (kindly provided by A. Olsen, Lawrence Livermore National Laboratory, Livermore, CA) was isolated and digested with restriction endonucleases Sau3A and Csp6I, ligated with the library primer 5′-ACT TGAGCTCGAGTATCCATGAACA-3′, and PCR-amplified with the same primer as described previously (14,20).

Two-Dimensional EMSA

For two-dimensional EMSA (2D-EMSA), a pool of short DNA fragments of a 563-kb FXYD5-TZFP region of human chromosome 19 was radioactively labeled by PCR with the library primer and purified as described previously (21). The 2D-EMSA was performed generally as described (21), but instead of purified DNA binding protein, 2.5 µg Jurkat cell nuclear extract protein was added to the initial EMSA reaction.