We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-B family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-B family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5 end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-B binding was also found at genes whose expression was regulated by tumor necrosis factor ␣, a known inducer of NF-B-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor ␣. Many of these latter genes are either known to be activated by NF-B under other conditions or are consistent with NF-B's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-B binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.U nderstanding the targets regulated by transcription factors and where they bind relative to these targets in an unbiased fashion in mammalian cells is highly desirable. We and others have developed a procedure for mapping in vivo targets of transcription factors by chromatin immunoprecipitation (ChIP) with antibodies to a transcription factor of interest to isolate protein-bound DNA, followed by probing a microarray containing genomic DNA sequences with the immunoprecipitated DNA (ChIP chip) (1-3). This approach was first used successfully in yeast and has more recently been used in a limited fashion to identify transcription factor binding sites in mammalian cells (4-6). However, a large-scale, unbiased global analysis of the distribution of mammalian transcription factor binding sites along large genomic regions has not been previously explored.In this study we employ a microarray containing the entire nonrepetitive sequence of chromosome 22 to determine the chromosome-wide binding profile for the transcription factor NF-B. The NF-B͞Rel family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including immune and inflammatory responses (7,8), and the activation pathway has been studied extensively over the last two decades (9, 10). Numerous NF-B target genes have also been identified; however, it remains unclear how many of these are direct targets of the transcription factor (11).There are five mammalian NF-B family members (p50, p52, RelA͞p65, RelB, and c-rel), all of which function as homo-or heterodimers. The different dimers exhibit varying binding aff...

A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A) + RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22.

The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5 ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans.The cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a key transcription factor that stimulates the expression of numerous genes in response to growth factors, hormones, neurotransmitters, ion fluxes, and stress signals. The CREB signaling pathway is activated by extracellular ligands that bind to cell surface receptors. Various intracellular second messengers then relay signals through kinase pathways to the nuclear resident CREB. Upon induction of the pathway, CREB is phosphorylated and activated at Ser-133. At least four types of kinases have been proposed to phosphorylate this residue: cAMP-dependent protein kinase, multiple mitogenactivated protein kinases (MAPKs), ribosome S6 kinase, Ca 2ϩ -and calmodulin-dependent kinases (CAMKs), and possibly Akt (reviewed in reference 22). Phosphorylation of CREB at Ser-133 leads to the recruitment of CREB binding protein (CBP) or its paralog p300 and subsequent transcriptional activation. A number of CREB targets have been identified, mostly through studies of individual genes (17, 26, 32). However, a full range of targets has not been explored. Given CREB's role in many important cellular processes, it is important to identify as many potential targets as possible.CREB functions either by itself or with the related family members CREM and ATF1; CREB, CREM, and ATF1 homoand heterodimerize through basic leucine zipper (bZIP) domains (27,40). A number of studies indicate that CREB is constitutively bound to chromatin even in the absence of agonists. CREB usually binds to cAMP response elements (CREs) near TATA boxes (26). However, not all DNA binding regi...

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