Straightforward protocol for allele-specific chromatin conformation capture.

A key advance in our understanding of gene regulation came with the finding that the genome undergoes three-dimensional nuclear folding in a genetically determined process. This 3D conformation directly influences the association between enhancers and their target promoters. This complex interplay has been proven to be essential for gene regulation, and genetic variants affecting this process have been associated to human diseases. The development of new technologies that quantify these DNA interactions represented a revolution in the field. High throughput techniques like HiC provide a general picture of chromatin topology. However, they often lack resolution to evidence subtle effects that single nucleotide polymorphisms exert over the contacts between cis-regulatory regions and target promoters. Here we propose a cost-efficient approach to perform allele-specific chromatin conformation analysis. As a proof of concept, we analyzed the impact of a common deletion mapping between SIRPB1 promoter and one of its downstream enhancers.

Assay for transposase-accessible chromatin and circularized chromosome conformation capture, two methods to explore the regulatory landscapes of genes in zebrafish.

Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome.

SIRPB1 copy-number polymorphism as candidate quantitative trait locus for impulsive-disinhibited personality.

Impulsive-disinhibited personality (IDP) is a behavioral trait mainly characterized by seeking immediate gratification at the expense of more enduring or long-term gains. This trait has a major role in the development of several disinhibitory behaviors and syndromes, including psychopathy, attention-deficit and hyperactivity disorder, cluster-B personality disorders, criminality and alcoholism. Available data consistently support a strong heritable component, accounting for 30-60% of the observed variance in personality traits. A genome-wide analysis of copy-number variants was designed to identify novel genetic pathways associated with the IDP trait, using a series of 261 male participants with maximized opposite IDP scores. Quantitative trait locus analysis of candidate copy-number variants (CNVs) was conducted across the entire IDP continuum. Functional effects of associated variants were evaluated in zebrafish embryos. A common CNV mapping to the immune-related gene SIRPB1 was significantly associated with IDP scores in a dose-dependent manner (ß=-0.172, P<0.017). Expression quantitative trait locus analysis of the critical region revealed higher SIRPB1 mRNA levels associated with the haplotype containing the deleted allele (P<0.0007). Epigenetic marks highlighted the presence of two potential insulators within the deleted region, confirmed by functional assays in zebrafish embryos, which suggests that SIRPB1 expression rates are affected by the presence/absence of the insulator regions. Upregulation of SIRPB1 has been described in prefrontal cortex of patients with schizophrenia, providing a link between SIRPB1 and diseases involving disinhibition and failure to control impulsivity. We propose SIRPB1 as a novel candidate gene to account for phenotypic differences observed in the IDP trait.

The 14q22.2 colorectal cancer variant rs4444235 shows cis-acting regulation of BMP4.

Common genetic variation at human 14q22.2 tagged by rs4444235 is significantly associated with colorectal cancer (CRC) risk. Re-sequencing was used to comprehensively annotate the 17kb region of strong linkage disequilibrium encompassing rs4444235. Through bioinformatic analyses using H3K4Me1, H3K4Me3, and DNase-I hypersensitivity chromatin signatures and evolutionary conservation we identified seven candidate disease-causing single-nucleotide polymorphisms mapping to six regions within the 17-kb region predicted to have regulatory potential. Reporter gene studies of these regions demonstrated that the element to which rs4444235 maps acts as an allele-specific transcriptional enhancer. Allele-specific expression studies in CRC cell lines heterozygous for rs4444235 showed significantly increased expression of bone morphogenetic protein-4 (BMP4) associated with the risk allele (P<0.001). These data provide evidence for a functional basis for the non-coding risk variant rs4444235 at 14q22.2 and emphasizes the importance of genetic variation in the BMP pathway genes as determinants of CRC risk.

Xiro-1 controls mesoderm patterning by repressing bmp-4 expression in the Spemann organizer.

The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro-1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim-1. Our results also indicate that Xiro-1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro-1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro-1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro-1 works as a repressor of bmp-4 transcription and that its effect on organizer development is dependent on BMP-4 activity. We propose that the previously observed down regulation of bmp-4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro-1 described here. Thus, Xiro-1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp-4 transcription.

The Iroquois family of genes: from body building to neural patterning.

The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.

A novel function for the Xslug gene: control of dorsal mesendoderm development by repressing BMP-4.

The Snail family of genes comprise a group of transcription factors with characteristic zinc finger motifs. One of the members of this family is the Slug gene. Slug has been implicated in the development of neural crest in chick and Xenopus by antisense loss of function experiments. Here, we have generated functional derivatives of Xslug by constructing cDNAs that encode the Xslug protein fused with the transactivation domain of the virus-derived VP16 activator or with the repressor domain of the Drosophila Engrailed protein. Our results suggest that Xslug normally functions as a transcriptional repressor and that Xslug-VP16 behaves as a dominant negative of Xslug. In the present work, we confirm and extend previous results that suggest that Xslug has an important function in neural crest development, by controlling its own transcription. In addition we have uncovered a new function for Xslug. We show that Xslug is expressed in the dorsal mesendoderm at the beginning of gastrulation, where is it able to upregulate the expression of dorsal genes. On the other hand when Xslug is expressed outside of the organizer it represses the expression of ventral genes. Our results indicate that this effect on mesodermal patterning depends on BMP activity, showing that Xslug can directly control the transcription of BMP-4.

XTIF2, a Xenopus homologue of the human transcription intermediary factor, is required for a nuclear receptor pathway that also interacts with CBP to suppress Brachyury and XMyoD.

Ligand-bound nuclear receptors (NRs) recruit cofactors such as members of the p160 family and CREB-binding protein (CBP) to activate transcription. We have cloned the Xenopus homologue of the human transcription intermediary factor 2 (TIF2), a member of the p160 family of cofactors. Xenopus TIF2 (XTIF2) mRNA is expressed homogeneously during late blastula-early gastrula stages and later becomes highly expressed in the notochord. To study the function of XTIF2 during development, we have used two dominant negative constructs, one encompassing the NR-binding domain and the other the CBP interacting region of XTIF2. Overexpression of the XTIF2 dominant negative mRNAs causes ectopic expression of Xenopus Brachyury (Xbra) and MyoD in all tissue layers. Moreover, ectopic expression of the dominant negative construct that contains the CBP-binding region produces strong phenotypes at hatching stage such as loss of head structures, shortened trunks and open blastopores, which can be rescued by XTIF2 coexpression. These observed defects are due, at least in part, to repression of dorsal mesoderm and endoderm genes. Our data suggest the existence of a NR pathway that requires XTIF2 and CBP to repress Xbra and XMyoD.

Xenopus brain factor-2 controls mesoderm, forebrain and neural crest development.

The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurula forebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.

Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate.

The Drosophila homeoproteins Ara and Caup are members of a combination of factors (prepattern) that control the highly localized expression of the proneural genes achaete and scute. We have identified two Xenopus homologs of ara and caup, Xiro1 and Xiro2. Similarly to their Drosophila counterparts, they control the expression of proneural genes and, probably as a consequence, the size of the neural plate. Moreover, Xiro1 and Xiro2 are themselves controlled by noggin and retinoic acid and, similarly to ara and caup, they are overexpressed by expression in Xenopus embryos of the Drosophila cubitus interruptus gene. These and other findings suggest the conservation of at least part of the genetic cascade that regulates proneural genes, and the existence in vertebrates of a prepattern of factors important to control the differentiation of the neural plate.

Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system.

In Drosophila the decision processes between the neural and epidermal fate for equipotent ectodermal cells depend on the activity of proneural genes. Members of the Drosophila Iroquois-Complex (Iro-C) positively regulate the activity of certain proneural AS-C genes during the formation of external sensory organs. We have identified and characterized three mouse Iroquois-related genes: Irx1, -2 and -3, which have a homeodomain very similar to that of the Drosophila Iro-C genes. The sequence similarity implies that these three genes represent a separate homeobox family. All three genes are expressed with distinct spatio/temporal patterns during early mouse embryogenesis. These patterns implicate them in a number of embryonic developmental processes: the A/P and D/V patterning of specific regions of the central nervous system (CNS), and regionalization of the otic vesicle, branchial epithelium and limbs.

araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing.

The homeo box prepattern genes araucan (ara) and caupolican (caup) are coexpressed near the anterior-posterior (AP) compartment border of the developing Drosophila wing in two symmetrical patches located one at each side of the dorsoventral (DV) compartment border. ara-caup expression at these patches is necessary for the specification of the prospective vein L3 and associated sensory organs through the transcriptional activation, in smaller overlapping domains, of rhomboid/veinlet and the proneural genes achaete and scute. We show that ara-caup expression at those patches is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus (Ci) protein in anterior cells near to the AP compartment border. The high levels of Ci activate decapentaplegic (dpp) expression, and, together, Ci and Dpp positively control ara-caup. The posterior border of the patches is apparently defined by repression by engrailed. Wingless accumulation at the DV border sets, also by repression, the gap between the two patches. Thus, ara and caup integrate the inputs of genes effecting the primary subdivisions of the wing disc into compartments to define two smaller territories. These in turn help create the even smaller domains of rhomboid/veinlet and achaete-scute expression.

iroquois: a prepattern gene that controls the formation of bristles on the thorax of Drosophila.

We have isolated a Drosophila mutant where the lateral parts of the notum are completely naked, leaving unaffected a median stripe of hairs. This mutation, iroquois (iro), defines a new gene which maps at 69D. We show that, in the presumptive lateral notum of mutant discs, sense organ precursor cells fail to form and the proneural gene scute is not expressed. The expression of a reporter gene inserted near iro suggests that iro itself is massively expressed in this region of the disc. We propose that iro is a prepattern gene essential to activate the expression of scute in the regions of the disc that will form the lateral notum.

Araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes.

In Drosophila imaginal wing discs, the achaete-scute (ac-sc) proneural genes and rhomboid (veinlet) are expressed in highly resolved patterns that prefigure the positions of sensory organs and wing veins, respectively. It is thought that these patterns are generated by a combination of factors (a prepattern) regulating these genes. We provide evidence for the existence of this prepattern by identifying two of its factors, Araucan and Caupolican. They are members of a novel family of homeoproteins, with homologs in vertebrates. Araucan and Caupolican, present in domains of the imaginal discs larger than those expressing ac-sc and rhomboid, are necessary for expression of these genes in the overlapping domains. Araucan and Caupolican appear to be positive, direct regulators of ac-sc.

Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs.

The pattern of bristles and other sensory organs on the adult cuticle of Drosophila is prefigured in the imaginal discs by the pattern of expression of the proneural achaete (ac) and scute (sc) genes, two members of the ac-sc complex (AS-C). These genes are simultaneously expressed by groups of cells (the proneural clusters) located at constant positions in discs. Their products (transcription factors of the basic-helix-loop-helix family) allow cells to become sensory organ mother cells (SMCs), a fate normally realized by only one or a few cells per cluster. Here we show that the highly complex pattern of proneural clusters is constructed piecemeal, by the action on ac and sc of site-specific, enhancer-like elements distributed along most of the AS-C (approximately 90 kb). Fragments of AS-C DNA containing these enhancers drive reporter lacZ genes in only one or a few proneural clusters. This expression is independent of the ac and sc endogenous genes, indicating that the enhancers respond to local combinations of factors (prepattern). We show further that the cross-activation between ac and sc, discovered by means of transgenes containing either ac or sc promoter fragments linked to lacZ and thought to explain the almost identical patterns of ac and sc expression, does not occur detectably between the endogenous ac and sc genes in most proneural clusters. Our data indicate that coexpression is accomplished by activation of both ac and sc by the same set of position-specific enhancers.

Inactivation of ornithine decarboxylase by intermediates of tyrosinase-catalyzed reaction.

1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway. 2. The inactivation was dependent on the substrate used (dihydroxybenzylamine > L-3,4-dihydroxyphenylalanine > L-tyrosine) and on the concentration of intermediate produced rather than on the rate of formation. 3. Sulfhydryl compounds (dithiothreitol and glutathione) or quinone-reducing agents (ascorbic acid) prevented the inactivation of ODC; L-ornithine, but not other amino acids, also protected partially ODC. The results suggest that different cysteine residues in ODC molecule are implicated in the inactivatory event. 4. When 14C-labeled catechols were used, numerous polypeptides resulted labeled, showing that the reactive quinones formed as intermediates in the process of melanin biosynthesis bind covalently to many cellular proteins.

Different turnover of rat fetal and placental ornithine decarboxylases.

The half-lives of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) have been studied in fetuses and placentas from 18-day-pregnant rats. While the turnover of fetal and placental SAMDC were slightly different (t1/2 = 38 and 75 min, respectively) the half-lives of fetal and placental ODC differed markedly. T1/2 of fetal ODC was 15 min, similar to other mammalian ODCs, but placental ODC showed a relatively high half-life, about 160 min. According to that, placental ODC was more resistant than the fetal enzyme to in vivo hyperthermic treatment (40 degrees C, 1 h). Our results suggest that the degradative mechanisms for ODC in rat placenta could be regulated differently to those in other mammalian tissues.