Novel interaction partners of the chromatin remodeler CHD7, a protein mutated in CHARGE syndrome

Zusammenfassung CHARGE Syndrom, ein autosomal dominant vererbtes Malformationssyndrom wird durch Mutationen im Chromodomänen Helikase DNA bindenden protein 7 (CHD7) Gen hervorgerufen. In 10% der „typischen“ und 40-50% der „atypischen“ CHARGE Patienten findet sich keine Mutation im CHD7 Gen und somit bleibt die Ursache der Symptomatik in diesen Fällen unklar. CHD7 ist ein nukleär lokalisierter chromatin remodeler, der in großen Multiproteinkomplexen detektiert werden konnte und die Expression verschiedener Gene reguliert. Die Charakterisierung von CHD7 Interaktionspartnern ist möglicherweise hilfreich um die Pathogenese des CHARGE Syndroms zu verstehen. Für einige andere genetisch bedingte Erkrankungen konnte gezeigt werden, dass Mutationen in Interaktionspartnern zu demselben oder einem ähnlichen Krankheitsbild führen. Daher vermuten wir, dass CHD7 Interaktionspartner gute Kandidaten sind, die möglicherweise im mutierten Zustand ebenfalls zum CHARGE Syndrom führen. Im ersten Teil dieser Arbeit wurde CHD8 als ein Interaktionspartner von CHD7 identifiziert. Die Interaktion des CHD7 Teilstückes (Aminosäuren: 1593-2178) mit einem CHD8 Teilstück (Aminosäuren: 1789-2302) wurde mittels verschiedener molekularer Techniken validiert. Zusätzlich konnten wir zeigen, dass die Interaktion im Nuckleoplasma stattfindet. Darüber hinaus wurde der Einfluss von 3 in der Literatur beschriebenen Mutationen (p.His2096Arg, p.Val2102Ile und p.Gly2108Arg) und einer neu identifizierten (p.Trp2091Arg) CHD7 missense Mutation auf die CHD7-CHD8 Interaktion mittels Yeast two hybrid (Y2H) und Co-Immunopräzipitation (Co-IP) untersucht. Während wir mittels Y2H zeigen konnten, dass die CHD7-CHD8 Interaktion durch die CHD7 missense Mutationen p.Trp2091Arg, p.His2096Arg und p.Gly2108Arg aufgehoben wird, konnte dieser Effekt mittels Co-IP nicht nachgewiesen werden. Daher vermuten wir, dass CHD7 und CHD8 einerseits direkt miteinander interagieren (gezeigt durch die direkten Y2H Experimente) und zusätzlich indirekt über sog. Linker Proteine, die zusammen mit CHD7 und CHD8 einen großen Proteinkomplex formen (Erklärung für die Co-IP Ergebnisse). Zusätzlich haben wir CHD7 negative CHARGE Patienten auf Mutationen im CHD8 Gen untersucht. Im zweiten Teil der Doktorarbeit zeigen wir die Ergebnisse der SILAC und Massenspektrometrie Analyse, bei der wir das bisher uncharakterisierte Protein FAM124B als eine Komponente eines CHD7-CHD8 enthaltenden Komplexes identifizierten. Die Interaktion mit dem CHD8 Teilstück (Aminosäuren 1789-2302) wurde mittels direktem Y2H und Co-IPs validiert, während eine Interaktion mit dem CHD7 Teilstück (Aminosäuren 1593-2178) nur mittels Co-IP bestätigt werden konnte, wodurch gezeigt wurde, dass FAM124B nicht direkt mit der o.g. CHD7 Region interagiert. Zusätzlich erfolgte eine Charakterisierung von FAM124B. Wir konnten zeigen, dass FAM124B, wie CHD7 und CHD8 im Zellkern lokalisiert ist. Fam124B wird bei der Maus in den Organen, die beim CHARGE Syndrom betroffen sind expremiert. Eine starke Expression konnte in dem sich entwickelnden Gehirn, Herzen, Lunge und im Rückenmark von E12.5 Mauseembryonen nachgewiesen werden. Kürzlich konnte gezeigt werden, dass Mutationen in CHD8 Autismus (ASD) und Entwicklungsstörungen des Nervensystems (NDD) bewirken können. Zusammenfassend, weisen unsere Daten darauf hin, dass das bisher uncharakterisierte Protein FAM124B eine sehr wichtige Funktion in der Embryonalentwicklung hat und möglicherweise an der Pathogenese des CHARGE Syndroms und ASD/NDD beteiligt ist. Um eine mögliche Rolle von FAM124B an der Pathogenese des CHARGE Syndroms und Neurocristopathien zu analysieren, planen wir die Herrunterregulierung von Fam124B in Xenopus laevis um anschließend den Einfluss von Fam124B auf Gene, die an der Neuralleistenzellformation beteiligt sind zu testen. Die Generierung eines Fam124B knockout Mausmodels und die weitere Charakterisierung von CHD7 Interaktionspartnern werden zur Erlangung tiefgreifender Kenntnisse des molekularen Mechanismus des CHARGE Syndroms und Autismus Erkrankungen hilfreich sein

Genetic analysis of mitochondrial ND5 gene of siberian ibex (Capra Sibirica, Pallas, 1776) population in Mongolia

The Siberian ibex (Capra sibirica) from Central Asia is believed to be the most ancient species of the genus Capra. In Mongolia, it is distributed in the areas of Mongolian Altai, Gobi-Altai, Dzungaria, Altai, Khan Khuhii, Khoridal Saridag and Ulaan Taiga as well as in the desert and semi-desert steppe zones of Dundgobi and Dornogobi aimags (provinces). In the current study, we investigated the mitochondrial ND5 gene fragments of the Siberian ibex population from different parts of Mongolia. Nine haplotypes, including 6 shared and 3 unique haplotypes were identified among these populations. Furthermore, Tajima’s statistics and Fu’s statistics did not reveal significant positive value across the population, indicating population decline and balancing selection.In the phylogenetic tree by 9 haplotypes, no separated clusters were generated. In addition, nucleotide diversity was 0.015, haplotype diversity was 0.86 and the average number of differences in nucleotides was 8.2 in the overall population. These results suggest that genetic diversity across all the populations was low, while haplotype diversity and the average number of differences in nucleotides were high

Genetic comparison of Altai and Gobi argali sheep (Ovis ammon) populations using mitochondrial and microsatellite markers: Implication on conservation

Argali sheep is an ungulate, which inhabits the north, west, south and central regions of Mongolia. There are two major populations (Altai and Gobi) in Mongolia, but their taxonomic classification as subspecies is often disputed among researchers. Furthermore, there is no recent study about the population genetic structure of argali sheep in Mongolia. In the present study, we have investigated genetic diversity and difference between Altai and Gobi argali populations using mitochondrial control region hyper variable segment (HVS) sequence (598bp) and 3 microsatellite markers. Mitochondrial HVS haplotype analysis showed high haplotype diversity (0.982±0.012) and low nucleotide diversity (0.02589). In microsatellite analysis, total of 9 alleles were found across all loci while mean Ho were 0.59±0.13 for Altai and 0.53±0.1 for Gobi populations, indicating low allelic diversity with moderate heterozygosity. Neighbor-joining tree separated haplotypes into two clusters, Altai and Gobi population, implying distinct genetic difference between the two subspecies. Additionally, Pairwise FST and Kimura-2 parameter showed 0.127 and 0.0413±0.0068, respectively. These genetic distance analyses hinted genetic difference between Altai and Gobi populations are in subspecies level. In summary, mitochondrial HVS and microsatellite analysis demonstrated that Altai and Gobi populations had low genetic diversity but might be genetically distinct from each other in subspecies level, suggesting conservation should be separately managed

Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex

BACKGROUND: Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry. PRINCIPLE FINDINGS: The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues. CONCLUSION: Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders

Western blot and immunocytochemistry of endogenous and overexpressed FAM124B.

<p>(<b>A</b>) Western blot analysis on protein isolated from untransfected HeLa cells (endogenous FAM124B) and HeLa cells overexpressing the FAM124B-1,3-cmyc-tag fusion protein. Lane 1: Immunoblotting of the nuclear cell fraction of untransfected HeLa cells using the FAM124B antibody. The predicted size of human endogenous FAM124B-1,3 is approximately 51 kDa. However, we observed a band of approximately 57 kDa (labeled by an asterisk). We hypothesize that this band could be endogenous FAM124B and the larger size is possibly due to posttranslational modifications of the endogenous protein. Lane 2: Immunoblotting of FAM124B-1,3-pCMV-cmyc overexpressed HeLa total cell lysate using the rabbit anti-FAM124B antibody revealed a prominent band of 51 kDa, the calculated size of human full length FAM124B variant 1 in fusion with a c-myc-tag and the band observed in untransfected HeLa cells. Lane 3: Immunoblotting of the same HeLa cell lysate as shown in lane 2 using anti-c-Myc shows the 51 kDa band corresponding to the overexpressed FAM124B. Lane 4: Marker. Protein quality was controlled using mouse anti-HSC70 producing a 70 kDa band. (<b>B</b>) Immunofluorescence staining using the rabbit anti-Fam124B antibody on HeLa cells transiently transfected with the plasmid FAM124B-1,3-pCMV-cmyc (FAM124B transcript variant 1 fused to an c-myc-tag). Due to the transient transfection not all cells overexpressed FAM124B. Transfected cells show a bright red signal in the nucleus demonstrating a nuclear distribution. The weaker signal in the cell nuclei of the untransfected surrounding cells could possibly reflect an endogenous FAM124B expression, which we detected by RT-PCR and mass spectrometry. (<b>C</b>) Immunofluorescence staining using the anti-c-Myc antibody on the same HeLa cells transiently transfected with the plasmid FAM124B-1,3-pCMV-cmyc showing a bright green signal in the nucleus of the transfected cells. No signal could be observed in the surrounding untransfected cells. (<b>D</b>) Cell nuclei were stained with DAPI (blue). (<b>E</b>) Overlay of B,C and D. (<b>F</b>) Immunofluorescence staining using the rabbit anti-Fam124B antibody on untransfected HeLa cells revealed a weak nuclear signal as it was observed in the surrounding untransfected cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052640#pone-0052640-g005" target="_blank">Figure 5B</a>). (<b>G</b>) Immunofluorescence staining using the anti-c-Myc antibody on untransfected cells reveals no signal, as expected. (<b>H</b>) Staining of cell nuclei with DAPI (<b>I</b>) overlay of F,G and H. B,C,D,E: Scale bar = 40 µm. F,G,H,I: Scale bar = 30 µm.</p

Fam124B expression in the mouse central nervous system.

<p>(<b>A</b>) Overview, (<b>B</b>) Thalamic nuclei, (<b>C</b>) Hippocampus, (<b>D</b>) Caudate Putamen, (<b>E</b>) Cerebellum. CC = Corpus Callosum, Hipp = Hippocampus, CA1-3 = Cornu Ammonis areas, DG = dentate gyrus, CP = Caudate Putamen, adt = anterior dorsal thalamic nucleus, dg = granual layer of dentate gyrus, lv = lateral ventricle, F = fornix, mdt = mediodorsal thalamic nucleus, IIIv = third ventricle with choroid plexus, vpl = ventral posterior thalamic nucleus, lateral part, ML = molecular layer of cerebellum, PL = purkinje cell layer of cerebellum, GL = granular layer of cerebellum, Scale bar = 100 µm.</p

Schematic overview of FAM124B, CHD7 and CHD8 and the constructs used.

<p>In humans, there are two transcript variants of FAM124B. Transcript variant 1 codes for a protein with 455 amino acids (NP_001116251.1), while transcript variant 2 leads to a shorter protein product with 272 amino acids (NP_079061.2). Both variants have the first 244 amino acids in common. All used FAM124B variant 1 and 2 constructs are full length constructs. Bioinformatic analysis of the amino acid sequence of FAM124B failed to identify any known functional domain, while CHD7 and CHD8 consist of two N-terminal chromodomains (Chromo), followed by a SWI2/SNF2-like ATPase/helicase domain (Helicase/ATPase), three conserved regions (CR1-3), a SANT domain and two BRK domains. The black lines mark the regions cloned into the Yeast two hybrid (pGBKT7, pGADT7) and Co-IP (pCMV-HA, pCMV-cmyc) vectors.</p

Co-immunoprecipitation of FAM124B with a part of CHD7 and CHD8.

<p>HeLa cells were co-transfected with either the CHD7-CR1-3-pCMV-HA (amino acids 1593-2178, NP_060250.2) plasmid and FAM124B-1,3-pCMV-cmyc or FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1) or with CHD8-pCMV-cmyc (amino acids 1789-2302, NP_065971.2) and FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1). (<b>A</b>) Using the anti-CHD8 (abcam, ab84527) or the anti-CHD7 (abcam, ab31824) antibody for precipitation, we detected with the anti-HA antibody (Roche) an approximately 51 kDa band corresponding to the estimated size of FAM124B transcript variant 1. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. (<b>B</b>) Reciprocal immunoprecipitation with anti-cmyc antibody (precipitating FAM124B transcript variant 1), and detection with the anti-CHD7 antibody lead to a specific band ∼70 kDa, the estimated size for the CHD7 part fused to the HA-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. (<b>C</b>) Reciprocal experiment with anti-HA antibody (precipitating FAM124B transcript variant 1) and detection with the anti-CHD8 antibody detected a specific band ∼68 kDa, the estimated size for the CHD8 part fused to the cmyc-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. (<b>D, E, F</b>) The same experimental procedure was performed for FAM124B transcript variant 2, demonstrating a specific interaction of FAM124B transcript variant 2 with the CHD7 and CHD8 part as well. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control.</p

In situ hybridization of Fam124B on mouse brain cryosections in comparison with immunostaining.

<p>(<b>A</b>) Immunostaining of a cortex section, (<b>B</b>) In situ hybridization (ISH) of a cortex section (<b>C</b>) Sense control of ISH. (<b>D</b>) Immunostaining of a hippocampus section, (<b>E</b>) In situ hybridization of a hippocampus section (<b>F</b>) Sense control of ISH. DG = dentate gyrus. Scale bar = 100 µm.</p

Yeast two hybrid assay.

<p>(<b>A</b>) Direct yeast two hybrid experiment with the constructs FAM124B-1,3-pGADT7 (full length transcript variant 1) and CHD7-CR1-3-pGBKT7 (amino acids 1591-2181, NP_060250.2) demonstrating no direct interaction between FAM124B transcript variant 1 and the CHD7 part, while (<b>B</b>) Direct yeast two hybrid experiment with the constructs FAM124B-1,3-pGADT7 (full length transcript variant 1) and CHD8-pGBKT7 (amino acids 1789–2302, NP_065971.2) shows a direct interaction. The same experiments were performed for FAM124B transcript variant 2. (<b>C</b>) Direct yeast two hybrid experiment with the constructs FAM124B-1,2-pGADT7 (full length transcript variant 2) and CHD7-CR1-3-pGBKT7 (amino acids 1591-2181, NP_060250.2). (<b>D</b>) Direct yeast two hybrid experiment with the constructs FAM124B-1,2-pGADT7 (full length transcript variant 2) and CHD8-pGBKT7 (amino acids 1789–2302, NP_065971.2). FAM124B transcript variant 2 interacts directly with the CHD8 part, while no direct interaction with the CHD7 part could be observed.</p