Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting

Abstract

A differentially methylated region (DMR) and endoderm-specific enhancers, located upstream and downstream of the mouse H19 gene, respectively, are known to be essential for the reciprocal imprinting of Igf2 and H19. To explain the same imprinting patterns in non-endodermal tissues, additional enhancers have been hypothesized. We determined and compared the sequences of human and mouse H19 over 40 kb and identified 10 evolutionarily conserved downstream segments, 2 of which were coincident with the known enhancers. Reporter assays in transgenic mice showed that 5 of the other 8 segments functioned as enhancers in specific mesodermal and/or ectodermal tissues. We also identified a conserved 39-bp element that appeared repeatedly within the DMR and formed complexes with specific nuclear factors. Binding of one of the factors was inhibited when the target sequence contained methylated CpGs. These complexes may contribute to the presumed boundary function of the unmethylated DMR, which is proposed to insulate maternal Igf2 from the enhancers. Our results demonstrate that comparative genomic sequencing is highly efficient in identifying regulatory elements.

Figure 1

Comparison of the human and mouse H19 region sequences. (A) Map of the Igf2/H19 region. The region analyzed is indicated by a horizontal line below the map. (B) Dot matrix alignment. (Top) Lower magnification of the H19 region. The homology plot program DNASIS v3.0 (Hitachi Software Engineering) was used to align the mouse (x axis) and human (y axis) H19 sequences with a criterion of 22 matching bases in a window of 30. Brackets with arrows in the matrix and bars between the map and matrix indicate conserved segments (CS1–CS10). (Inset) Higher magnification of the CS1–CS4 region. The GenBank sequences used are as follows: mouse, AF049091 (bases 1–41680); human, AF087017 (bases 1–40558) and AC004556 (bases 82896–88682).

Figure 2

Expression of the lacZ reporter gene driven by the conserved downstream segments in transgenic mice. (A) Structure of the reporter construct. Each transgene construct was made by insertion of one of the conserved segments into plasmid pIZ (Hatano et al. 1998). (B) Representative patterns of lacZ staining in 12- to 12.5-dpc embryos. CS1 transgenes reproducibly stained the ganglionic placodes of the facial nerve (near the first branchial groove) and the inferior edge of the maxillary prominences. CS3 transgenes were expressed in the sclerotome, and CS4 transgenes in the liver and the epithelial layer of the gut. CS5 directed lacZ expression in the neural tube floor plate as well as in the ectoderm of the limb buds. Both CS6 and CS9 transgenes expressed lacZ in the myotome and the rib primordia. CS7 transgenes were expressed in the mesenchyme condensations at the bases of the limb buds. The expression patterns were confirmed by histological examinations.

Figure 3

Evolutionarily conserved 39-bp sequence elements in the upstream DMR of H19. (A) Sequence alignment of the elements conserved in human (hcs1–hcs6), mouse (mcs1–mcs5) and rat (rcs1–rcs5). The bases identical to the consensus sequence (bottom) are shaded. The highly conserved core of the consensus sequence is underlined. Nucleotide positions (in parentheses) follow numbering schemes for AF087017 (human), AF049091 (mouse), and AF043428 (rat). (B) Map of the upstream DMR of human, mouse, and rat H19. (Stippled boxes) G-rich repeat region found in rodents; (hatched boxes) two types of the human 400-bp repeat sequences; (open arrowheads) locations of the conserved 39-bp sequences. B, BamHI; R, EcoRI.

Figure 4

Formation of complexes between the conserved upstream elements and nuclear factors revealed by EMSA. (A) The five mouse elements, mcs1–mcs5, form complexes with specific nuclear proteins in embryos and ES cells. Nuclear extracts prepared from 12.5-dpc mouse embryos and ES cells were incubated with ³²P-labeled mcs1–mcs5 oligonucleotide duplexes. The affinity between the factors and elements differed greatly. The patterns obtained with the two different extracts appeared identical except that the ES cell extracts gave one or two additional faint bands with higher mobilities. (B) Lack of cross-competition between mcs1, mcs4, SP1, and AP2 duplexes. (C) Methylation of CpG in mcs1 reduces complex formation. Effects of methylation were not evident for mcs4.

Figure 4

Formation of complexes between the conserved upstream elements and nuclear factors revealed by EMSA. (A) The five mouse elements, mcs1–mcs5, form complexes with specific nuclear proteins in embryos and ES cells. Nuclear extracts prepared from 12.5-dpc mouse embryos and ES cells were incubated with ³²P-labeled mcs1–mcs5 oligonucleotide duplexes. The affinity between the factors and elements differed greatly. The patterns obtained with the two different extracts appeared identical except that the ES cell extracts gave one or two additional faint bands with higher mobilities. (B) Lack of cross-competition between mcs1, mcs4, SP1, and AP2 duplexes. (C) Methylation of CpG in mcs1 reduces complex formation. Effects of methylation were not evident for mcs4.