Computational approach to identifying tissue-specific CRM. (A) The algorithm is based on the following steps: (1) identification of tissue-specific genes that are highly or lowly expressed based on statistical analysis of microarray expression data of normal human tissues; (2) extraction of the corresponding promoter sequences from publicly available databases; (3) mapping transcription factor binding sites (TFBSs) to these promoters by using the TRANSFAC database and identification of the tissue-specific CRM using a differential distance matrix (DDM)/multidimensional scaling (MDS) approach⁵ ; and (4) searching the genomic context of the highly expressed genes for evolutionary conserved CRM. (B) Evolutionary conservation, nucleotide sequence, and TFBSs located within the 71-bp HS-CRM8 element from human SERPINA1 identified by the aforementioned algorithm. The TFBSs include binding sites for FOXA1 (blue), CEBP (yellow), HNF1 (light green), MyoD (purple), LEF-1 (dark green), and LEF-1/TCF (brown). Some of these TFBSs are partially overlapping. (C) Chromatin immunoprecipitation assay confirming the binding of FOXA1 and CEBP on HS-CRM8. Antibodies specific to FOXA1 and CEBP and polymerase chain reaction (PCR) primers specific for the corresponding TFBS were used. In particular, PCR primers were designed to amplify a region within the vector corresponding to HS-CRM8 (that binds FOXA1 and CEBP), an untranscribed region on chromosome 6 was used as negative control (–). Binding events per 10³ cells (mean + standard deviation) were determined for each of the corresponding primer pairs. Significant differences compared with the negative control were indicated (Student t test, *P ≤ .05). (D) Confocal microscopy of different organs of mice injected with AAV9-HS-CRM8-TTR-GFP (5 × 10¹¹ vg/mouse; n = 4) with 4′,6 diamidino-2-phenylindole nuclear staining (top panels). A representative confocal scan is shown. Noninjected mice were used as controls (bottom panels). Pictures were taken at ×20 magnification.