Bruno Pichon

Nuclear targeting of proteins: how many different signals?

The nuclear import of proteins into the cell nucleus involves the recognition of a nuclear localization signal sequence, borne by the protein to be transported, by complex molecules called importins, that will subsequently mediate the crossing over of the nuclear envelope. The most frequently encountered signal sequence is made up of short stretches of basic amino acid residues and is recognized by importins alpha and/or beta. Other signal sequences have been described, and some have been shown to mediate the association with importins other than importin alpha or beta. Recently, approaches have been developed that allow the cloning, on a functional basis, of sequences able to specify the nuclear localization of proteins. A variety of peptidic motifs of limited size which do not contain previously described signal sequences were isolated in such assays. It reveals that the spectrum of sequences that are able to target a protein to the cell nucleus may be wider than currently expected. It will probably also lead to the identification of novel target sequences for importins and will demonstrate the implication of additional members of this family of proteins in nuclear transport.

Implementation of genomic arrays in prenatal diagnosis: The Belgian approach to meet the challenges

After their successful introduction in postnatal testing, genome-wide arrays are now rapidly replacing conventional karyotyping in prenatal diagnostics. While previous studies have demonstrated the advantages of this method, we are confronted with difficulties regarding the technology and the ethical dilemmas inherent to genomic arrays. These include indication for testing, array design, interpretation of variants and how to deal with variants of unknown significance and incidental findings. The experiences with these issues reported in the literature are most often from single centres. Here, we report on a national consensus approach how microarray is implemented in all genetic centres in Belgium. These recommendations are subjected to constant re-evaluation based on our growing experience and can serve as a useful tool for those involved in prenatal diagnosis.

A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly

Primary microcephaly (microcephalia vera) is a developmental abnormality resulting in a small brain, with mental retardation. It is usually transmitted as an autosomal recessive trait, and six loci have been reported to date. We analyzed a translocation breakpoint previously reported in a patient with apparently sporadic primary microcephaly, at 1q31, where locus MCPH5 maps. The patient was lost to follow-up, and we sampled a maternal aunt who carried the familial translocation. FISH analyses showed that the insert of BAC clone RP11-32D17 spanned the breakpoint. The breakpoint was further located within a fragment of this insert corresponding to intron 17 of the ASPM gene, resulting in a predicted transcript truncated of more than half of its coding sequence. It is very likely that the proband carried a second ASPM mutation in trans, but he was not available for sampling and hence we could not confirm this hypothesis. Our observation adds to the mutation spectrum of ASPM in primary microcephaly, and is to our knowledge the second example of a constitutional, reciprocal translocation responsible for a bona fide autosomal recessive phenotype.

XHRT-1, a hairy and Enhancer of split related gene with expression in floor plate and hypochord during early Xenopus embryogenesis.

Abstract. We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.

Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the Orange domain of the hairy-related transcription factor HRT1/Hey1 in xenopus and mouse

Hairy‐related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix‐loop‐helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer‐of‐split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two‐hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity. Developmental Dynamics 228:716–725, 2003.

A Familial Heterozygous Null Mutation of MET in Autism Spectrum Disorder

Autism spectrum disorder (ASD) results from interactions of genetic and environmental factors. The MET proto‐oncogene has been identified as a candidate gene for autism susceptibility, and is implicated in neurodevelopment and social brain circuitry. Here, we describe the first case of a familial mutation of MET, consisting of an interstitial genomic deletion removing exons 12 through 15, causing a frameshift and premature stop codon, with evidence of nonsense‐mediated mRNA decay. On the other allele, patients carried the C allele of the MET promoter rs1858830 polymorphism, known to decrease MET expression and previously associated with autism susceptibility. The heterozygous mutation was associated with autism in one patient, and language and social impairment in a sibling. Our observations delineate the phenotypic spectrum associated with a clearly defined, very likely complete loss of function mutation of MET. Incomplete penetrance in this family was consistent with MET as a partial susceptibility gene for ASD. Implication of MET in normal and pathological brain development opens new perspectives for understanding the pathophysiology of autism and for eventual therapeutical clues. Autism Res 2014, 7: 617–622.

Expression of a transactivation-deficient form of thyroid transcription factor I decreases the activity of co-transfected thyroglobulin and thyroperoxidase promoters

Thyroid transcription factor I (TTF‐1) plays a critical role in thyroid organogenesis and in the control of expression of several thyroid‐specific genes, like those coding for thyroglobulin and thyroperoxidase. We have expressed the isolated DNA‐binding homeodomain of TTF‐1 in cultured thyroid cells by transient transfection. A specific reduction in the activity of cotransfected thyroglobulin and thyroperoxidase promoters was observed in the presence of the isolated TTF‐1 homeodomain, as compared to their activity measured in the presence of a mutated homeodomain unable to bind DNA. The activity of the SV40 early promoter, used as a control, was only marginally affected in these experiments. The transactivation‐deficient form of TTF‐1 described here may thus be used for investigating other cellular processes that are dependent on TTF‐1 transcriptional activity.

A canonical nerve growth factor-induced gene-B response element appears not to be involved in the cyclic adenosine monophosphate-dependent expression of differentiation in thyrocytes.

The expression of transcriptionally active nerve growth factor-induced gene-B (NGFI-B) is rapidly induced in thyroid follicular cells in response to cAMP stimulation. As the transcription of thyrocyte-specific genes is controlled by the cAMP cascade, we have investigated a possible involvement of NGFI-B in this control. Recombinant adenoviruses driving the expression of either the intact NGFI-B protein or a truncated form of it that lacks the capacity to transactivate a NBRE-dependent promoter, were used to infect dog thyrocytes maintained in primary culture. Northern blot analysis of total RNA from infected cells revealed that the expression of NGFI-B was not sufficient to induce a significant accumulation of specific transcripts (thyroglobulin, thyroperoxidase, sodium-iodide symporter) in unstimulated thyrocytes. The overproduction of the transcriptionally inactive form of NGFI-B in thyrocytes maintained in the presence of forskolin after infection did not impair the accumulation of the thyroid-specific transcripts. These data show that NGFI-B does not control the expression of differentiation in thyrocytes by acting through a canonical NBRE. As a consequence, we must consider that either the expression of NGFI-B in cAMP-stimulated thyrocytes is not critically linked to the expression of differentiation or that NGFI-B is implicated in a regulatory mechanism which differs from its known action at the level of a NBRE.

Activation by thyroid stimulating hormone of nerve growth factor-induced gene-B expression in thyrocytes in culture: relation with proliferation and specific gene expression

Nerve growth factor-induced gene-B (NGFI-B) is an immediate early gene first found as a part of the PC12 cell response to NGF (Milbrandt, J., Science 238 (1987) 797-799). We have previously reported that NGFI-B mRNA is strongly upregulated by thyroid-stimulating hormone (TSH) in dog thyrocytes in culture (Pichon et al., Endocrinology 137 (1996) 4691-4698). In this study, we have analyzed the regulation of NGFI-B mRNA expression by a variety of agents acting on thyrocytes proliferation and/or differentiation. We show that: (1) the induction of NGFI-B mRNA is stronger after stimulation of the cAMP cascade, but it is not restricted to this signaling pathway; (2) the powerful mitogens for thyroid cells EGF and HGF have little or no effect on NGFI-B mRNA induction; (3) NGFI-B mRNA is induced by anisomycin at a subinhibitory concentration for protein synthesis, and is superinduced by the combination of TSH and anisomycin; this treatment decreases the TSH-induced proliferation levels, but does not inhibit the induction of some differentiation markers; and (4) both in dog and in pig thyrocytes, NGFI-B mRNA induction is observed after a variety of treatments stimulating differentiation, but without proliferative effects. Our results therefore suggest that NGFI-B mRNA induction might not be related to TSH-induced thyrocyte proliferation, but could participate in the differentiation program triggered by TSH.

DNA methylation and gene activity: towards the end of the debate?

On average, the DNA of the vertebrate genome ccmtairrs about 5% of 5-methylcytosine. This so-called “fifth base” shows, however, a non-random distribution in the DNA. Moreover, methylated cytosine content of the genome may change during the development of the individual. For example, increased levels of DNA methylation are observed in early embryonic cells, as compared to adult tissues, whereas extra-embryonic lineages exhibit a striking undermethylation of their D:NA (Jaenisch and Jahner, 1984; Sanford et al., 1985). In vertebrates, methylated cytosines are almost exclusively found in CG dinucleotide motifs, the C residues on both strands of the DNA being methylated (Gruenbaum et al., 1981). The CG dinucleotide itself appears clearly under-represented in most of the genome, except in small areas, the “HTF islands” (Bird, 19861, which make up about 1% of the total DNA. These sequences exhibit a higher content in G and C bases, as compared to the bulk DNA, and contain essentially unmethylated CG dinucleotides. The paucity of the CG motif in the rest of the DNA is thought to be a consequence of cytosine methylation itself, the 5methylcytosine residue showing a propensity to deaminate to form a thymidine. This relative instability of the “fifth base” may also contribute significantly to the incidence of genetic diseases by causing deleterious mutations (Cooper and Youssoufian, 1988; Jones et al., 1992), despite the fact that a specific repair pathway, which counteracts the mutagenic effects of the deamination, exists in mammalian cells (Wiebauer and Jiricny, 1989). The pattern of cytosine methylation in the genome is dictated by as yet unknown signals. Evidence for the existence of a control specific for methylation of DNA sequences exists: (i) the establishment of allele-specific patterns of methylation, also known as “genomic im-