Issues related to central counterparty clearing: opening remarks

Banks and banking, Central

Transcriptional regulation of Hox genes during hindbrain development.

Hox genes play a crucial role in patterning the anteroposterior axis. Therefore, it is important to understand the mechanisms regulating their expression. One way of identifying regulatory regions is to compare related genomic sequences in order to find conserved elements. Previous work in transgenic mice has defined the elements required to recapitulate Hoxa2 expression in rhombomeres 3 and 5. However, the mechanisms regulating expression of Hoxa2 in rhombomeres 2 and 4 were unknown. In this study, I demonstrate that a highly conserved region in the intron of Hoxa2 contains the control elements directing rhombomere 4 expression. Further, I show that the rhombomere 2 enhancer is located in the second exon of Hoxa2. Due to genome-wide duplication events, the number of Hox genes increased during vertebrate evolution. In the pufferfish, this led to the presence of two Hoxa2 genes, Hoxa2(a) and Hoxa2(b). The two co-paralogous genes show differential expression. I compared the control regions of these two genes and identified subtle changes in their cw-regulatory regions. Using chick electroporation, I show that several changes in these elements are responsible for the differential expression. Hoxbl has a broad expression pattern in the hindbrain at early stages. This expression becomes restricted to rhombomere 4 during later development. I show that Krox20 binds to a highly conserved repressor region and that removal of this element in transgenic constructs leads to an expansion of reporter expression into rhombomeres 3 and 5. Finally, I show that Hoxbl expression in the second branchial arch is repressed by a mammalian-specific repressor element. This work has shed important insight into the mechanisms and factors that modulte expression of Hoxa2 and Hoxbl during hindbrain development

Forward to the special issue on Hox/Tale transcription factors in development and disease

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102217/1/dvdy24098.pd

An ultraconserved Hox–Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4

The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints

Characterization of the Regulatory Region of the Zebrafish Prep1.1 Gene: Analogies to the Promoter of the Human PREP1

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5′ upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to −1.8, possibly −5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the −5.0 Kb promoter. A transgenic fish expressing GFP under the −1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the −3 to −5 Kb of the human upstream region

Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons

Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity

Hox Paralog Group 2 Genes Control the Migration of Mouse Pontine Neurons through Slit-Robo Signaling

The pontine neurons (PN) represent a major source of mossy fiber projections to the cerebellum. During mouse hindbrain development, PN migrate tangentially and sequentially along both the anteroposterior (AP) and dorsoventral (DV) axes. Unlike DV migration, which is controlled by the Netrin-1/Dcc attractive pathway, little is known about the molecular mechanisms guiding PN migration along the AP axis. Here, we show that Hoxa2 and Hoxb2 are required both intrinsically and extrinsically to maintain normal AP migration of subsets of PN, by preventing their premature ventral attraction towards the midline. Moreover, the migration defects observed in Hoxa2 and Hoxb2 mutant mice were phenocopied in compound Robo1;Robo2, Slit1;Slit2, and Robo2;Slit2 knockout animals, indicating that these guidance molecules act downstream of Hox genes to control PN migration. Indeed, using chromatin immunoprecipitation assays, we further demonstrated that Robo2 is a direct target of Hoxa2 in vivo and that maintenance of high Robo and Slit expression levels was impaired in Hoxa2 mutant mice. Lastly, the analysis of Phox2b-deficient mice indicated that the facial motor nucleus is a major Slit signaling source required to prevent premature ventral migration of PN. These findings provide novel insights into the molecular control of neuronal migration from transcription factor to regulation of guidance receptor and ligand expression. Specifically, they address the question of how exposure to multiple guidance cues along the AP and DV axes is regulated at the transcriptional level and in turn translated into stereotyped migratory responses during tangential migration of neurons in the developing mammalian brain

Mutations in the Polycomb Group Gene polyhomeotic Lead to Epithelial Instability in both the Ovary and Wing Imaginal Disc in Drosophila

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional polyhomeotic targets are implicated in this phenomenon.Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of polyhomeotic sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors

De Novo Genesis of Enhancers in Vertebrates

Whole genome duplication in teleost fish reveals that a few changes in non-regulatory genomic sequences are a source for generating new enhancers