Epigenetic regulation of gene expression is essential in many biological processes, and its deregulation contributes to pathology including tumor formation. We used an image-based cell assay that measures the induction of a silenced GFP-estrogen receptor reporter to identify novel classes of small molecules involved in the regulation of gene expression. Using this Locus Derepression assay, we queried 283,122 compounds by quantitative high-throughput screening evaluating compounds at multiple concentrations. After confirmation and independent validation, the Locus Derepression assay identified 19 small molecules as new actives that induce the GFP message over 2-fold. Viability assays demonstrated that 17 of these actives have anti-proliferative activity, and two of them show selectivity for cancer versus patient-matched normal cells and cause unique changes in gene expression patterns in cancer cells by altering histone marks. Hence, these compounds represent chemical tools for understanding the molecular mechanisms of epigenetic control of transcription and for modulating cell growth pathways.