Once conserved non-coding sequences (CNSs) regions have been identified, the next step is to evaluate their function in biological experiments. There are various techniques to assess regulatory function.

DNASE I Hypersensitivity: Hypersensitive sites are regions at which local nucleosome organization has been altered from that of surrounding areas. They are often occupied by or show increased accessibility to transcription factors and other DNA-binding proteins. Ideally, bioinformatic and hypersensitve site analyses should extend for 50-100 kb in either direction from the gene, and intronic regions of neighboring genes should be included in the analysis, as they may contain important regulatory elements.

Once hypersensitive site mapping has been completed for a cell type of interest, it is worthwhile to extend it to other cell types and stimulation conditions. CNS regions that show increased hypersensitivity in stimulated cells may correspond to inducible enhancers whose function can be tested in standard reporter assays.

Developmental and cell lineage analyses are also particularly informative. For example, precursor naive T cells show DNase I hypersensitivity only at hypersensitive sites 3 and IV, located 5′ and 3′ of IL4. Th1 cells, which derive from the same naive precursors but have silenced IL4, continue to display hypersensitive sites 3 and IV. However, neither of these sites is apparent in fibroblasts, an unrelated cell type that has also silenced IL4. A testable hypothesis following from these data is that hypersenstivie sites 3 and IV have distinct, cell type-specific functions: in Th1 cells, the sites may participate in silencing of the cytokines genes, whereas in naive T cells, the sites may be responsible for the poised state of the locus, for which the exact stimulation conditions determine whether gene activation or silencing prevails.

Targeted Disruption: One of the most reliable means of assessing in vivo function is targeted disruption of putative regulatory regions. Deletions and mutations of regulatory regions can be done either in the native chromosomal context or in large bacterial or yeast artificial chromosome transgenes. Deletion of positive regulatory elements such as enhancers will result in decreased gene expression. Deletion of negative regulatory elements such as silencers will lead to increased gene expression in cells that either normally express or normally silence the gene. Loss of an insulator element may lead to inappropriate expression of either the gene in question or a neighboring gene in an irrelevant cell type.

It is important to note that for important loci (e.g., IL4) for which the ability to survive attack by pathogens is crucial for reproductive fitness and survival, deletion of individual hypersensitive sites may have only a partial effect, most likely because evolutionary pressures have imposed functional redundancy such that more than one regulatory region participates in gene activation or silencing. In such cases, multiple mutations may be needed to produce strong effects.

Reporter Assays: A variety of reproter assays using cell lines or transgenic animals have been used to asses whether putative enhancer, silencer and insulator elements influence gene expression from target promoters.

Chromatin immunoprecipitation: As DNA-binding proteins associated with a regulatory region are likely to recruit DNA and histone modifying enzymes, a CNS involved in regulating gene expression is likely to be a focus for differential histone modifications or differential DNA methylation in cells that express or do not express the gene. This can be evaluated by chromatin immunoprecipitation with antibodies to modified histone tails and by restriction digestion with methylation-sensitive enzymes.