Protein – DNA – Interactions Laboratory
Team: Frank Kaiser (leader), Ronja Hollstein (PhD student), Juliane Eckhold (technical assistent)
For many years, it was thought that the gene expression is solely regulated by binding of several transcription factors to the promoter region which is located just 5´ of the translation start site. This view has dramatically changed within the past years. Physical interactions influencing gene expression have been shown between distinct regions that can be separated by hundreds of kilo base pairs (kb) or even located on different chromosomes. While only about 1.5% of the human genome is protein-coding, a biochemical function was assigned to up to 80% of the genome.1 For instance, >8% of the genome is occupied by transcription factor binding and >50% is enriched for histone modifications. Of note, the transcription factor binding is non-randomly distributed across the genome. Co-localization of transcription factors combined with other features such as specific histone modifications can be used to predict the regulatory function and status of selected genomic region.
To investigate the putative functional relevance of newly identified variants affecting regulatory elements (such as promoters, enhancers, 3’-/5’-untranslated regions), a broad spectrum of different molecular approaches (e.g. reporter gene assays, quantitative PCR, chromatin immunoprecipitation assay (ChIP), electromobility shift assays (EMSAs) are available within the laboratory and have successfully been applied to functionally characterize selected genomic variants and their relevance for gene expression. 2-7
One important emerging key contributor to genomic function is the spatial organization of DNA in the cell nucleus as we have described recently.8 It is becoming increasingly apparent that long-range control of gene expression is facilitated by chromatin looping interactions76. Active and inactive genes are engaged in many long-range intra-chromosomal interactions and can also form inter-chromosomal contacts77. First described in 2002, chromosome conformation capture (3C) technology provides an opportunity to identify presumably regulatory DNA-DNA interactions in a genome-wide fashion78. The technology relies on the idea that digestion and religation of fixed chromatin in cells, followed by the quantification of ligation junctions, allows for the determination of DNA contact frequencies and insights into chromosome topology79. While with 3C technology interacting sequences are detected by PCR of linear fragments and thus limited to a ‘one-versus-one’ approach (with one primer in the ‘viewpoint’ (selected region of interest) and the other primer in the candidate interacting region), 4C technology involves a circularization step (circular chromosome conformation capture, 4C)80. This enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners allowing for a ‘one-versus-all’ strategy.
ENCODE_Project_Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 2012;489:57-74.
Bach AS, Derocq D, Laurent-Matha V, Montcourrier P, Sebti S, Orsetti B, Theillet C, Gongora C, Pattingre S, Ibing E, Roger P, Linares LK, Reinheckel T, Meurice G, Kaiser FJ, Gespach C, Liaudet-Coopman E. (2015) Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.Oncotarget. 2015 Sep 29;6(29):28084-103
Schmidt K, Kaiser FJ, Erdmann J, Wit Cd. (2015) Two polymorphisms in the Cx40 promoter are associated with hypertension and left ventricular hypertrophy preferentially in men. Clin Exp Hypertens. 2015;37(7):580-6.
Erogullari A, Hollstein R, Seibler P, Braunholz D, Koschmidder E, Depping R, Eckhold J, Lohnau T, Gillessen-Kaesbach G, Grünewald A, Rakovic A, Lohmann K, Kaiser FJ.(2014) THAP1, the gene mutated in DYT6 dystonia, autoregulates its own expression. Biochim Biophys Acta. 2014 Nov;1839(11):1196-204.
Miller CL, Haas U, Diaz R, Leeper NJ, Kundu RK, Patlolla B, Assimes TL, Kaiser FJ, Perisic L, Hedin U, Maegdefessel L, Schunkert H, Erdmann J, Quertermous T, Sczakiel G.(2014) Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation. PLoS Genet. 2014 Mar 27;10(3):e1004263.
Sowa AK, Kaiser FJ, Eckhold J, Kessler T, Aherrahrou R, Wrobel S, Kaczmarek PM, Doehring L, Schunkert H, Erdmann J, Aherrahrou Z. (2913) Functional interaction of osteogenic transcription factors Runx2 and Vdr in transcriptional regulation of Opn during soft tissue calcification. Am J Pathol. 2013 Jul;183(1):60-8.
Deardorff MA, Bando M, Nakato R, Watrin E, Itoh T, Minamino M, Saitoh K, Komata M, Katou Y, Clark D, Cole KE, De Baere E, Decroos C, Di Donato N, Ernst S, Francey LJ, Gyftodimou Y, Hirashima K, Hullings M, Ishikawa Y, Jaulin C, Kaur M, Kiyono T, Lombardi PM, Magnaghi-Jaulin L, Mortier GR, Nozaki N, Petersen MB, Seimiya H, Siu VM, Suzuki Y, Takagaki K, Wilde JJ, Willems PJ, Prigent C, Gillessen-Kaesbach G, Christianson DW, Kaiser FJ, Jackson LG, Hirota T, Krantz ID, Shirahige K. (2013) HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature. 2012 Sep 13;489(7415):313-7.
Watrin E, Kaiser FJ, Wendt KS. (2016) Gene regulation and chromatin organization: relevance of cohesin mutations to human disease. Curr Opin Genet Dev. 2016 Jan 25;37:59-66.