Given the importance of transcription factors in facilitating vital aspects of cell biology, mutations in -or aberrant regulation oftranscription factors have been associated with human disease. The identification of inhibitors or activators of transcription factors will therefore not only illuminate the signaling pathways that regulate them, but could also identify targets that may prove to be better drug targets than transcription factors themselves, or whose 2-Pyridylethylamine dihydrochloride inhibition may provide a more selective therapeutic effect. We chose to screen for inhibitors of NF-��B, a family of transcription factors that in mammals plays a central role in regulating immune responses, development, cell proliferation, and survival. They form dimers and are normally kept inactive in the cytoplasm. Activation of a wide variety of receptors, including antigen receptors, patternrecognition receptors and cytokine receptors leads to translocation of NF-��B dimers into the nucleus. Here the dimers bind to DNA ��B sites in promoters and enhancers of target genes. Activation of NF-��B needs to be tightly controlled and rapidly curtailed following the initial stimulus to prevent uncontrolled tissue damage and/or disease. Here we performed the first reporter screen in KBM7 cells to identify constitutive inhibitors of NF-��B. The identification of CYLD, a known negative regulator of NF-��B, demonstrates the utility of using human haploid cells to dissect a variety of biological processes. All screens in human haploid cells performed to date have relied on intrinsic phenotypes, such as sensitivity to A 839977 toxins or protein surface expression, both of which can be easily observed at a cellular level. To provide a clear phenotypic readout for abrogation of NF-��B inhibitor function -and thus improper activation of NF-��B-we generated a NF-��B reporter cell line. We transduced KBM7 cells, which are haploid for all chromosomes but chromosome 8, with a reporter construct that contains a NF-��B transcriptional response element and a minimum cytomegalovirus promoter upstream of the blasticidin S resistance gene from Bacillus cereus. Thus, insertional inactivation of genes that normally repress activation of NF-��B would render the reporter cells resistant to blasticidin and provide an easy means to distinguish them from wild-type cells. To ensure that the selected clonal reporter cell line had intact NF-��B regulation, we stimulated both KBM7 cells and the NF-��B reporter cell line with TNF. We saw that both cells displayed similar degradation kinetics. The selected clonal reporter cell line survived in the presence of blasticidin only when stimulated with NF-��B activators, demonstrating that the reporter functioned properly. The NF-��B reporter cell line was then mutagenized with a retroviral gene-trap vector, using an established protocol that generally yields a library containing mutations in approximately 98% of genes expressed in KBM7 cells. Mutagenized NF-��B reporter cells were exposed to blasticidin and the survivors were pooled and expanded. The selected mutant population was markedly more resistant to blasticidin than the parental reporter cell line and wild-type KBM7 cells in the absence of any stimulus, suggesting that the survivors contain mutations that cause constitutive activation of NF-��B. To identify the mutations in the selected mutant population, genomic DNA was harvested from the survivors. The DNA sequences that flank gene-trap insertion sites were amplified, sequenced in parallel, and mapped to the human genome.