Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recuited in vertebrates. Here we have identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, we provide evidence that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates.

The early transcriptional hierarchy that subdivides the vertebrate hindbrain into seven to eight segments, the rhombomeres (r1–r8), is largely unknown. The Kreisler (MafB, Krml1, Val) gene is earliest gene expressed in an r5/r6-restricted manner and is essential for r5 and r6 development. We have identified the S5 regulatory element that directs early Kreisler expression in the future r5/r6 domain in 0–10 somite stage embryos. variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1β/LF-3B) is transiently expressed in the r5/r6 domain of 0–10 somite stage embryos and a vHNF1binding site within this element is essential but not sufficient for r5/r6-specific expression. Thus, early inductive events that initiate Kreisler expression are clearly distinct from later-acting ones that modulate its expression levels. This site and some of the surrounding sequences are evolutionarily conserved in the genomic DNA upstream of the Kreisler gene among species as divergent as mouse, humans, and chickens. This provides the first evidence of a direct requirement for vHNF1 in initiation of Kreisler expression, suggests that the role of vHNF1 is evolutionarily conserved, and indicates that vHNF1 collaborates with other transcription factors, which independently bind to the S5 regulatory region, to establish the r5/r6 domain.

The embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1–r8). The Kreisler ( Krml1/MafB/val ) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreisler enu ( kr enu ) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler‐dependent transcription. Comparison of kr enu /kr enu embryos with those homozygous for the classic kreisler ( kr ) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the “r5‐like” phenotype of kr enu /kr enu and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino ( val ) suggest that Kreisler performs many of its r5‐specific functions by associating with other proteins. By contrast, kr/kr and kr enu /kr enu mouse and val‐/‐ zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse kr enu /kr enu and zebrafish val‐/‐ embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr /+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development. Developmental Dynamics 227:134–142, 2003. © 2003 Wiley‐Liss, Inc.