Regulation by homeoproteins: a comparison of deformed-responsive elements.

Homeotic genes of Drosophila melanogaster encode transcription factors that specify segment identity by activating the appropriate set of target genes required to produce segment-specific characteristics. Advances in understanding target gene selection have been hampered by the lack of genes known to be directly regulated by the HOM-C proteins. Here we present evidence that the gene 1.28 is likely to be a direct target of Deformed in the maxillary segment. We identified a 664-bp Deformed Response Element (1.28 DRE) that directs maxillary-specific expression of a reporter gene in transgenic embryos. The 1.28 DRE contains in vitro binding sites for Deformed and DEAF-1. The Deformed binding sites do not have the consensus sequence for cooperative binding with the cofactor Extradenticle, and we do not detect cooperative binding to these sites, though we cannot rule out an independent role for Extradenticle. Removing the four Deformed binding sites renders the 1.28 DRE inactive in vivo, demonstrating that these sites are necessary for activation of this enhancer element, and supporting the proposition that 1.28 is activated by Deformed. We show that the DEAF-1 binding region is not required for enhancer function. Comparisons of the 1.28 DRE with other known Deformed-responsive enhancers indicate that there are multiple ways to construct Deformed Response Elements.

Cap 'n' collar B cooperates with a small Maf subunit to specify pharyngeal development and suppress deformed homeotic function in the Drosophila head.

The basic-leucine zipper protein Cap 'n' collar B (CncB) suppresses the segmental identity function of the Hox gene Deformed (Dfd) in the mandibular segment of Drosophila embryos. CncB is also required for proper development of intercalary, labral and mandibular structures. In this study, we provide evidence that the CncB-mediated suppression of Dfd requires the Drosophila homolog of the mammalian small Maf proteins, Maf-S, and that the suppression occurs even in the presence of high amounts of Dfd protein. Interestingly, the CncB/Maf-S suppressive effect can be partially reversed by overexpression of Homothorax (Hth), suggesting that Hth and Extradenticle proteins antagonize the effects of CncB/Maf-S on Dfd function in the mandibular segment. In embryos, multimers of simple CncB/Maf-S heterodimer sites are transcriptionally activated in response to CncB, and in tissue culture cells the amino-terminal domain of CncB acts as a strong transcriptional activation domain. There are no good matches to CncB/Maf binding consensus sites in the known elements that are activated in response to Dfd and repressed in a CncB-dependent fashion. This suggests that some of the suppressive effect of CncB/Maf-S proteins on Dfd protein function might be exerted indirectly, while some may be exerted by direct binding to as yet uncharacterized Dfd response elements. We also show that ectopic CncB is sufficient to transform ventral epidermis in the trunk into repetitive arrays of ventral pharynx. We compare the functions of CncB to those of its vertebrate and invertebrate homologs, p45 NF-E2, Nrf and Skn-1 proteins, and suggest that the pharynx selector function of CncB is highly conserved on some branches of the evolutionary tree.

Developmental patterning genes and their conserved functions: from model organisms to humans.

Molecular and genetic evidence accumulated during the past 20 years in the field of developmental biology indicates that different animals possess many common genetic systems for embryonic patterning. In this review we describe the conserved functions of such developmental patterning genes and their relevance for human pathological conditions. Special attention is given to the Hox genetic system, involved in establishing cell identities along the anterior-posterior axis of all higher metazoans. We also describe other conserved genetic systems, such as the involvement of Pax6 genes in eye development and the role of Nkx2.5-type proteins in heart development. Finally, we outline some fascinating problems at the forefront of the studies of developmental patterning genes and show how knowledge obtained from model genetic organisms such as Drosophila helps to explain normal human morphogenesis and the genetic basis of some birth defects.

A sequence motif distinct from Hox binding sites controls the specificity of a Hox response element.

Hox transcription factors, in combination with cofactors such as PBC proteins, provide diverse developmental fates to cells on the anteroposterior body axis of animal embryos. However, the mechanisms by which the different Hox proteins and their cofactors generate those diverse fates remain unclear. Recent findings have provided support for a model where the DNA binding sites that directly interact with Hox-PBC heterodimers determine which member of the Hox protein family occupies and thereby regulates a given target element. In the experiments reported here, we test the function of chimeric Hox response elements and, surprisingly, find evidence that runs counter to this view. A 21 bp cofactor binding sequence from an embryonic Deformed Hox response element, containing no Hox or Hox-PBC binding sites, was combined with single or multimeric sites that bind heterodimers of Labial-type Hox and PBC proteins. Normally, multimerized Labial-PBC binding sites are sufficient to trigger a Labial-specific activation response in either Drosophila or mouse embryos. Here we find that the 21 bp sequence element plays an important role in Deformed specificity, as it is capable of switching a Labial-PBC binding site/response element to a Deformed response element. Thus, cofactor binding sites that are separate and distinct from homeodomain binding sites can dictate the regulatory specificity of a Hox response element.

Monodactylous limbs and abnormal genitalia are associated with hemizygosity for the human 2q31 region that includes the HOXD cluster.

Vertebrates have four clusters of Hox genes (HoxA, HoxB, HoxC, and HoxD). A variety of expression and mutation studies indicate that posterior members of the HoxA and HoxD clusters play an important role in vertebrate limb development. In humans, mutations in HOXD13 have been associated with type II syndactyly or synpolydactyly, and, in HOXA13, with hand-foot-genital syndrome. We have investigated two unrelated children with a previously unreported pattern of severe developmental defects on the anterior-posterior (a-p) limb axis and in the genitalia, consisting of a single bone in the zeugopod, either monodactyly or oligodactyly in the autopod of all four limbs, and penoscrotal hypoplasia. Both children are heterozygous for a deletion that eliminates at least eight (HOXD3-HOXD13) of the nine genes in the HOXD cluster. We propose that the patients' phenotypes are due in part to haploinsufficiency for HOXD-cluster genes. This hypothesis is supported by the expression patterns of these genes in early vertebrate embryos. However, the involvement of additional genes in the region could explain the discordance, in severity, between these human phenotypes and the milder, non-polarized phenotypes present in mice hemizygous for HoxD cluster genes. These cases represent the first reported examples of deficiencies for an entire Hox cluster in vertebrates and suggest that the diploid dose of human HOXD genes is crucial for normal growth and patterning of the limbs along the anterior-posterior axis.

A cap 'n' collar protein isoform contains a selective Hox repressor function.

We have characterized a protein isoform (CncB) from the Drosophila cap 'n' collar locus that selectively represses cis-regulatory elements that are activated by the Hox protein Deformed. Of the three Cnc protein isoforms, CncB is expressed in a localized pattern in mandibular and labral cells of the head during mid-stages of embryogenesis. When CncB protein is absent or reduced, mandibular cells are homeotically transformed toward maxillary identities. This transformation is associated with persistent Deformed expression in anterior mandibular cells, since the Deformed autoactivation circuit is normally antagonized by CncB function in these cells. Heat-shock-induced ectopic expression of CncB in mid-stages of embryogenesis is sufficient to attenuate the activity of Dfd response elements in maxillary epidermal cells, but appears to have no effect in trunk epidermal cells on either the function or the response elements of other Hox proteins. CncB provides a mechanism to modulate the specificity of Hox morphogenetic outcomes, which results in an increase in the segmental diversity in the Drosophila head.

Prader-Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method.

The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered.