Hoxb1 Enhancer and Control of Rhombomere 4 Expression: Complex Interplay between PREP1-PBX1-HOXB1 Binding Sites

Abstract

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.Hox proteins belong to a large family of transcription factors that control cell identity, differentiation, and patterning in embryonic development. The ordered expression of these genes along the body axis is a critical aspect of regional identity (24, 37). For example, the functional identity of segmental units, rhombomeres (r), in the vertebrate hindbrain depends on the combinatorial activity of Hox proteins along the anteroposterior axis (reviewed in references 22, 24, 27, and 38). Members of the Hox protein family participate in a cascade that involves regulation of their spatial and temporal expression by direct auto-, para-, and/or cross-regulatory mechanisms. Initial expression of Hox genes in the hindbrain is regulated by a variety of transient inputs, including retinoic acid (RA) and fibroblast growth factors (FGFs) (2,21,35,36,48,51) along with several transcription factors, such as retinoic acid receptors, Kreisler, and Krox20 (33,34,44,54). Following this initial phase, direct cross talk and feedback circuits between the Hox genes are one of the important mechanisms that serve to maintain restricted segmental expression once the early cues are no longer functioning (17,29,32,40,49,50).

Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

“…These data are therefore in complete agreement with the inhibition of HoxB transcription by all conditions that block actin polymerization. The same complex is also likely present on the enhancer of HoxB1 (because Prep1 also regulates this gene; Ferretti et al, 2000Ferretti et al, , 2005. The nature of the ␤-actin-Prep1 interaction is still unknown.…”

“…RA therefore induces the recruitment of both sequencespecific DNA-binding elements (Prep1 at the very least, but more likely the Prep1-Pbx1 dimer or the Prep1-Pbx1-HoxB1 trimer; Ferretti et al, 2000Ferretti et al, , 2005, actin, and its polymerizing enzyme(s) N-WASP and RNAPII.…”

“…Unlike monomeric Prep1 and Pbx1, dimeric Prep1-Pbx1 complexes bind DNA and interact with HoxB1 to activate HoxB1 and HoxB2 transcription This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09 -02-0114) on May 28, 2009. (Berthelsen et al, 1998;Ryoo et al, 1999;Ferretti et al, 2000Ferretti et al, , 2005Huang et al, 2002).…”

“…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).…”

Molecular and cellular mechanisms of adipogenesis

“…Prep1, взаимодействуя с Pbx1, влияет на специфичность его связывания с ДНК, а также значительно повышает аффинность. Prep1 и Pbx1 совместно участвуют в процес-сах эмбриогенеза, в частности, они образуют с фактором Hoxb1 тройной комплекс, регулирующий экспрессию генов в эмбриогенезе [54].…”