Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

Abstract

Correct regulation of the segment.restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxbl will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxbl, Hoxal, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxbl homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxbl involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxbl in maintaining r4 identity may be mediated by the Hoxb2 gene. Segmentation in the developing vertebrate hindbrain generates repeated morphological units, termed rhombomeres. These segmental units are lineage-restricted cellular compartments that provide a means of allocating blocks of cells that have distinct properties (for review, see Lumsden 1990;Wilkinson 1993;Keynes and Krumlauf 1994). Underlying this cellular organization, the patterns of expression of a number of transcription factors, growth factors, tyrosine kinase receptors, and their ligands have boundaries of expression that are tightly linked to specific hindbrain segments (for review, see Keynes and Krumlauf 1994;Lumsden and Krumlauf 1996). Prominent among these are the Hox genes whose expression patterns form an ordered set of overlapping domains that correlate with their gene order along the Present addresses: ~Department

The Recruitment of SOX/OCT Complexes and the Differential Activity of HOXA1 and HOXB1 Modulate the Hoxb1Auto-regulatory Enhancer Function

“…This is adjacent to R2 and separated from R3 by 17 nucleotides. A similar combination of a TALE site and a HOX/PBX site is also found in an r4-restricted cross-regulatory enhancer from the Hoxb2 gene, except that the sites are separated by 8 base pairs (11,15). In the case of Hoxb2 there is no overlapping SOX/OCT site, but the TALE and HOX/PBX sites synergize in vitro and in vivo to allow the formation of a trimeric and transcriptionally active HOXB1-PBX1-MEIS/PREP complex required for enhancer activity in transgenic assays (10,11).…”

“…Transgenic analysis in mice has led to the identification of sequences that function as HOX target sites in vivo for the auto-, cross-, and para-regulatory interactions among Hox genes (6,(12)(13)(14)(15). Several of these in vivo target sites are composed of bipartite and overlapping HOX-and PBX-binding motifs (HOX/ PBC sites) and represent useful models for analyzing the function of HOX-containing transcriptional complexes.…”

Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1

“…Furthermore, it was shown that the YPWM motif of Hoxa5 is essential for the biological speci®city of this gene (Zhao et al, 1996). More recently, HOXB1 and PBX1 were shown to cooperatively increase the expression activity of a reporter construct containing a Hoxb1 autoregulatory element (Rocco et al, 1997), and that a bipartite HOX-PBX binding motif present in Hoxb2 is necessary for its regulation in rhombomere 4 (Maconochie et al, 1997).…”

Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

“…Several lines of evidence have demonstrated the importance of RA receptors (RARs) for patterning and basal expression of Hox genes in the vertebrate neural tube (Marshall et al, 1994(Marshall et al, , 1996Gould et al, 1998;Dupe et al, 1999). However, expression of anterior HoxB genes also depends on an auto-regulatory circuit involving the HoxB1 protein and the Prep1-Pbx1 complex (Marshall et al, 1996;Maconochie et al, 1997;Dupe et al, 1999;Jacobs et al, 1999;Ryoo et al, 1999;Ferretti et al, 2000Ferretti et al, , 2005Huang et al, 2002). Prep1 and Pbx1 are essential genes in embryonic development (Selleri et al, 2001;Waskiewicz et al, 2002;Deflorian et al, 2004;Penkov et al, 2005;Ferretti et al, 2006;Di Rosa et al, 2007).…”