Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

Ubx orchestrates tissue identity through regional and bidirectional changes to chromatin accessibility

Hox proteins are homeodomain transcription factors that diversify serially homologous segments along the animal body axis, as revealed by the classic bithorax phenotype of Drosophila melanogaster where mutations in Ultrabithorax (Ubx) transform the third thoracic segment into the likeness of the second thoracic segment. To specify segment identity we show that Ubx both increases and decreases chromatin accessibility, coinciding with its role as both an activator and repressor of transcription. Surprisingly, whether Ubx functions as an activator or repressor differs depending on the proximal-distal position in the segment and the availability of Hox cofactors. Ubx-mediated changes to chromatin accessibility positively and negatively impact the binding of Scalloped (Sd), a transcription factor that is required for appendage development in both segments. These findings reveal how a single Hox protein can modify complex gene regulatory networks to transform the identity of an entire tissue.

Genome-wide binding analyses of HOXB1 revealed a novel DNA binding motif associated with gene repression

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in ES cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1 and MEIS can bind to this motif. In vivo, this motif is sufficient to direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

Meis family proteins are required for hindbrain development in the zebrafish

Meis homeodomain proteins function as Hox-cofactors by binding Pbx and Hox proteins to form multimeric complexes that control transcription of genes involved in development and differentiation. It is not known what role Meis proteins play in these complexes, nor is it clear which Hox functions require Meis proteins in vivo. We now show that a divergent Meis family member, Prep1, acts as a Hox co-factor in zebrafish. This suggests that all Meis family members have at least one shared function and that this function must be carried out by a conserved domain. We proceed to show that the Meinox domain, an N-terminal conserved domain shown to mediate Pbx binding, is sufficient to provide Meis activity to a Pbx/Hox complex. We find that this activity is separable from Pbx binding and resides within the M1 subdomain. This finding also presents a rational strategy for interfering with Meis activity in vivo. We accomplish this by expressing the Pbx4/Lzr N-terminus, which sequesters Meis proteins in the cytoplasm away from the nuclear transcription complexes. Sequestering Meis proteins in the cytoplasm leads to extensive loss of rhombomere (r) 3- and r4-specific gene expression, as well as defective rhombomere boundary formation in this region. These changes in gene expression correlate with impaired neuronal differentiation in r3 and r4, e.g. the loss of r3-specific nV branchiomotor neurons and r4-specific Mauthner neurons. We conclude that Meis family proteins are essential for the specification of r3 and r4 of the hindbrain.