Biomedical subjects

Bas van Steensel

Publications and source records attributed to Bas van Steensel.

5 recordsLinked to original sources

PubMed2003

Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression.

Animals↗

PubMed2003

Genomewide analysis of Drosophila GAGA factor target genes reveals context-dependent DNA binding.

The association of sequence-specific DNA-binding factors with their cognate target sequences in vivo depends on the local molecular context, yet this context is poorly understood. To address this issue, we have performed genomewide mapping of in vivo target genes of Drosophila GAGA factor (GAF). The resulting list of approximately 250 target genes indicates that GAF regulates many cellular pathways. We applied unbiased motif-based regression analysis to identify the sequence context that determines GAF binding. Our results confirm that GAF selectively associates with (GA)(n) repeat elements in vivo. GAF binding occurs in upstream regulatory regions, but less in downstream regions. Surprisingly, GAF binds abundantly to introns but is virtually absent from exons, even though the density of (GA)(n) is roughly the same. Intron binding occurs equally frequently in last introns compared with first introns, suggesting that GAF may not only regulate transcription initiation, but possibly also elongation. We provide evidence for cooperative binding of GAF to closely spaced (GA)(n) elements and explain the lack of GAF binding to exons by the absence of such closely spaced GA repeats. Our approach for revealing determinants of context-dependent DNA binding will be applicable to many other transcription factors.

Amino Acid Motifs↗

PubMed2003

Epigenomic profiling using microarrays.

Genes occupy only a minor fraction of genomes such as ours; however, histone and nonhistone chromosomal proteins and methylated DNA bases are distributed over both genic and nongenic regions. These widespread "epigenomic" features can be mapped and characterized by alternative applications of the same microarray technologies that have been used for conventional transcriptional profiling. Here we describe diverse microarray-based strategies for profiling patterns of DNA methylation, DNA replication, DNA binding, and chromatin-associated proteins and histone modifications. The rapid progress that is being made in developing and applying epigenomic profiling methods and the increasing availability of microarrays mean that epigenomic profiling is likely to become a standard research tool for understanding chromatin structure and gene expression during development.

Animals↗

PubMed2002

Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing.

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes.

Animals↗

PubMed2002

Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3.

DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.

Arabidopsis↗