137. Identification and Functional Analysis of Evolutionarily Conserved Non-Coding Sequences in the Human 5q31 Cytokine Cluster Region 

Gabriela Cretu, Webb Miller, Catherine M. Brion, Jan-Fang Cheng, Christopher H. Martin, William Kimberly, Edward M. Rubin, and Kelly A. Frazer 
Genome Sciences Department, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS 84-171, Berkeley, CA 94720  
kelly@mhgc.lbl.gov 

The human 5q31 region chosen by the JGI for large scale sequencing is biologically interesting because it harbors a family of cytokine genes which are important regulators of the immune response. We previously annotated the 1 Mb Cytokine Gene Cluster Region on human 5q31 computationally and biologically resulting in the identification of 23 genes and the determination of their expression patterns. To further annotate this 1 Mb region we are currently identifying evolutionarily conserved non-coding sequences and attempting to determine their biological function. Since the interleukin loci in this region are likely to have arisen by ancient duplications of an ancestral gene, several intervals of this region may be paralogous with each other. To identify evolutionarily conserved non-coding elements we compared the potential human paralogous sequences in this 1 Mb region to each other as well as with their orthologous mouse chromosome 11 sequences. Several highly conserved non-coding sequences that potentially have biological function were identified. We have started functionally analyzing two of these non-coding elements: an 86 bp element located 5' of the human interleukin 13 (IL 13) gene which is 91% identical with another non-coding element in the human 5q31 region, and a 400 bp element located between the IL 4 and IL 13 genes which is 85% identical in humans and mice.  

To investigate the biological function of the 86 bp element upstream of IL 13 we have deleted it in a human 5q31 YAC and have used this manipulated YAC to generate transgenic mice. Production of human IL 4 and IL 13 is markedly reduced in mice harboring the mutated YAC lacking the 86 bp conserved element compared with the production of these human proteins in mice harboring a wild type YAC. These data suggest that the 86 bp conserved element is involved in regulating the expression of the human IL 4 and IL 13 genes. 

The biological role of the non-coding 400 bp element located between IL 4 and IL 13 is being investigated by several strategies. We determined by PCR amplification and sequence analysis that this 400 bp element is also highly conserved in the cow, dog, rabbit, pig, and rat which provides additional evidence indicating that this human-mouse conserved non-coding elememt is likely to be functionally important. To determine the biological role of this conserved element we are in the process of homozygously deleting it in mice and will determine how its absence affects the expression of the murine IL 4 and IL 13 genes. We are also performing comparative studies of human IL 4 and IL 13 expression in mice harboring a 5q31 YAC lacking the 400 bp conserved element with mice harboring a wild type 5q31 YAC. To be able to assay for small changes in the expression of the human IL 4 and IL 13 genes it is important to eliminate variation in their expression due to integration site of the mutated and wild type YAC in the mouse genome. To accomplish this we have surrounded the 400 bp conserved element on the human YAC with lox P sites and are using this YAC to generate transgenic mice. The founder mice will be mated with wild type mice and with mice expressing CRE recombinase. In this manner we plan to generate lines of mice that harbor at the same site of integration the human YAC lacking and containing the 400 bp element. By assaying for changes in the expression of the human IL 4 and IL 13 genes in these mice we hope to gain insight into the function of this highly conserved 400 bp non-coding element.