Protein Dimerization Domain Research Articles

Genome Sequencing to Discover Drivers of Clonal Expansion in Smoldering Multiple Myeloma

Background Better prediction models are required to identify patients with high-risk smoldering multiple myeloma (SMM) who can benefit from early intervention therapy before progression. Several studies have reported heterogeneity of genetic abnormalities found at the SMM stage, but whether any mutations are sufficient to cause, or instead prevent progression, remains an important open question. We leveraged whole-genome sequencing (WGS) in a large cohort of precursor patients, including repeated assessment over time, to discover drivers of disease growth and monitor malignant transformation to multiple myeloma (MM). Methods WGS was performed on tumor and normal cells from 141 untreated patients with SMM or Monoclonal Gammopathy of Undetermined Significance (MGUS) from the PCROWD study. Moreover, this dataset was combined with genomic data from the literature totaling 1,034 patients with WGS and/or exomes sampled across MM stages for comparative analysis and biomarker discovery. MutSig2CV, GISTIC2, and a novel method for structural variants were used to establish the list of candidate drivers across disease stages, with statistical power to detect myeloma drivers from points mutations found in ≥2% of patients. Longitudinal WGS data were analyzed with the PhylogicNDT suite of tools to characterize clonal competition in treated and untreated patients. Results Patients were followed for a median time of 20 months from the sample date (range: 3 to 78 months), during which 11 progressed. Patients evenly distributed across 20/2/20 stages: 44 Low-Risk (37%), 29 Intermediate-Risk (24%), 47 High-Risk (39%) SMM were combined with 20 MGUS, showing our method is reliable to perform WGS in lower disease burden settings. A validation cohort comprising 61 SMM with a median follow-up time of 36 months (range: 5 to 121), with 22 progressions was used to corroborate our findings. As significant drivers could be specifically enriched among indolent disease (i.e., protective alleles), we ran de novo driver mutation discovery across MM stages. In addition to well-characterized MM drivers ( KRAS, NRAS, etc.), 16 new candidate genes were found significantly mutated, including IKFZ3 (Aiolos), a transcription factor and direct target of degradation with lenalidomide therapy, harboring frameshift and stop-gain mutations in the protein dimerization domain which could affect complete differentiation of plasma cells. Mutants in KRAS found primarily at codons Q61, G12, G13, and A146, were found in all disease risk groups and associated with progression to overt myeloma (Odds Ratio: 6.1, CI95%: [1.4, 27], p=0.01, q<0.1). Next, we investigated genes targeted by copy-number abnormalities and found candidates in four regions with focal gains, including on chr(1q21), chr(8q24)( MYC), chr(3q26), and chr(16p13) encompassing BCMA. These genes have higher expression in patients with broad gain (e.g. BCMA: P=8.8E-3) or focal amplification (e.g. BCMA: P=6.2E-5), and therefore are likely to be underlying targets of the rearrangements driving cancer growth. Of note, BCMA antigen point mutations remained undetected in SMM, therefore patients with gain(16p) represent an attractive population for targeted therapy with anti-BCMA agents, in particular when the gain is clonal (n=3/8, 38%). In 15 patients, serial sampling of tumor cells allowed longitudinal WGS analyses and reconstruction of phylogenetic trees of disease clones (2 to 3 timepoints, 4 progressors, median 14 months). Maintenance of the major clone with subclonal structure evolving over time was confirmed in all patients, and we observed a steady increase in the cancer cell fraction of known MM mutations (del(13q), KRAS, DIS3 loss of function) in untreated patients. In one patient receiving early interventional treatment, a 70% reduction of M-spike concentration was achieved after four cycles of therapy; however, partial response was found in tandem with the selection of a high-risk del(1p) and of a KRAS activating G12S mutation, before increase and clinical progression to MM. Conclusion These results highlight the power of genomic profiling in MM for early detection, discovery of novel drivers, monitoring of clonal selection and transformation to malignant disease. We show SMM is not a simple genomically-mature disorder, but rather a dynamic state with competing subclones, which could be leveraged for therapeutic interventions.

SARS‐CoV‐2 ORF8 Accessory Protein Dimerization Domains and Protein‐Protein Host Interaction

As the world continues to fight the coronavirus pandemic caused by SARS‐CoV‐2, we are gaining valuable insights by comparing this virus to other human coronaviruses such as SARS‐CoV and MERS‐CoV that have also caused outbreaks. Coronaviruses infect many animal species, but disease severity varies between strains. Viral accessory proteins are generally implicated in increased infectivity, pathogenicity, and virulence. The accessory protein open reading frame 8 (ORF8), although not essential for viral replication, appears to play a critical role in disease severity by inhibiting multiple pathways of the immune response. Furthermore, the ORF8 gene is found within a highly variable portion of the genome, increasing the possibility of dangerous mutant forms arising. Structure‐based design of therapeutics holds great promise for effective targeting of such rapidly evolving threats. Thus, we designed physical models and computer representations of ORF8, based on the published structure 7JTL, to better evaluate the structural features responsible for viral pathogenicity. ORF8 exists as a homodimer held together by amino acid residues that interact through hydrophobic interactions, hydrogen bonding, a salt bridge, and a disulfide bond. These interactions occur in a region referred to as the covalent interface. Another interface, referred to as the noncovalent interface, exists between separate homodimers and results in oligomerization. The homodimers in the oligomer are held together by an intermolecular beta sheet that is stabilized by an extensive hydrophobic surface. These two dimerization interfaces are unique to SARS‐CoV‐2 and have been proposed to be involved in the protein's ability to evade and suppress the host immune response. The computer and physical representations allow for better visualization of the ORF8 structure to evaluate the role of the multimeric structure. ORF8 has been found to interact with various host proteins, including Interleukin‐17 Receptor A (IL17RA), a pro‐inflammatory cytokine. To further corroborate the existence of this interaction and to better understand the role oligomerization might play in pathogenicity, we assessed the possible interactions of ORF8 (7JTL) and IL17RA (5N9B) through theoretical modeling using available tools such as HDOCK. A better understanding of these inter‐subunit interactions may improve our understanding of the unique mechanism the virus uses to evade the immune system and may be instrumental in the development of effective therapeutics.

In silico comparative analysis of SARS-CoV-2 Nucleocapsid (N) protein using bioinformatics tools

The world has been encountered to one of the biggest pandemics that causing by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is placed in the Beta-CoV genus in the Coronaviridae family. N protein is one of the crucial structural proteins of SARS-CoV-2 that binds to the genome thereby generating helical ribonucleoprotein core. It is involved in viral transcription/replication, translation, and viral assembly after entering the host cell through interacting with host proteins. N protein sequences of SARS-CoV-2 and taxonomically related CoVs are examined using bioinformatics tools and approaches including sequence alignment, sequence and phylogenetic analyzes, and predicting of putative N-Glycosylation and phosphorylation positions and also predictions and comparative analyzes are performed on 3D structures of N proteins from SARS-CoV-2 related CoVs through using of some of applied bioinformatics analyzes. Results of mega BLAST search revealed that the most similar N protein sequence to SARS-CoV-2 is Bat-CoV RaTG13 N protein sequence in the taxonomically related CoVs. SARS-CoV-2 is grouped with SARS, pangolin, civet and bat CoVs (RATG13, SL ZC45 and SL ZXC21) in N protein, nucleotide and protein based ML phylogenetic trees. Some of SARS-CoV-2 N proteins were showed divergence from other SARS-CoV-2 N proteins analyzed due to amino acid substitutions detected in SARS-CoV-2 N proteins samples in phylogenetic trees. The highest amino acid substitutions were detected in Richmont/USA (QJA42209.1) and Greece (QIZ16579.1) samples, with 2 and 3 place substitutions, respectively. By domain analyzes, three domains were detected as Corona_nucleocora (Pfam), N terminal CoV RNA-binding domain (HAMAP) and C terminal N protein dimerization domain (HAMAP). Possible N-glycosylation positions of SARS-CoV-2 N protein were predicted at two positions. Assessments of possible serine, threonine and tyrosine phosphorylations were found to be at 100 positions, 34 of them were higher than 80% possibility. 3D structure analysis based on TM scores revealed that although the results of 3D structure analysis were shown consistency with the taxonomy of the CoVs, the 3D structures of SARS-CoV-2 N protein and taxonomically related CoVs were not at the same fold.

High level of fetal-globin reactivation by designed transcriptional activator-like effector

AbstractThe fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and β thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the Gγ and Aγ-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetal-globin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the γ-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of β-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the β-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetal-globin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.

Modular Thermal Control of Protein Dimerization.

Protein-protein interactions and protein localization are essential mechanisms of cellular signal transduction. The ability to externally control such interactions using chemical and optogenetic methods has facilitated biological research and provided components for the engineering of cell-based therapies and materials. However, chemical and optical methods are limited in their ability to provide spatiotemporal specificity in light-scattering tissues. To overcome these limitations, we present "thermomers", modular protein dimerization domains controlled with temperature-a form of energy that can be delivered to cells both globally and locally in a wide variety of in vitro and in vivo contexts. Thermomers are based on a sharply thermolabile coiled-coil protein, which we engineered to heterodimerize at a tunable transition temperature within the biocompatible range of 37-42 °C. When fused to other proteins, thermomers can reversibly control their association, as demonstrated via membrane localization in mammalian cells. This technology enables remote control of intracellular protein-protein interactions with a form of energy that can be delivered with spatiotemporal precision in a wide range of biological, therapeutic, and living material scenarios.

Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling

Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S50 values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a ‘mixed’ UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B′–C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic π–π stacking interactions, two S-aromatic (face) interactions, and the presence of ‘proline brackets’. The homology model further provides important insights into structure–function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.

Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Identification of novel Bach2 transcripts and protein isoforms through tagging analysis of retroviral integrations in B-cell lymphomas

BackgroundThe Bach2 gene functions as a transcriptional repressor in B-cells, showing high expression level only before the plasma cell stage. Several lines of evidence indicate that Bach2 is a B-cell specific tumor suppressor. We here address patterns of insertional mutagenesis and expression of Bach2 is a murine retroviral model of B-cell lymphoma induction.ResultsWe report that the Bach2 gene is a target of proviral integrations in B-cell lymphomas induced by murine leukemia virus. An alternative Bach2 promoter was identified within intron 2 and this promoter was activated in one of the tumors harboring proviral integration. The alternative promoter was active in both normal and tumor tissue and the tissue specificity of the two Bach2 promoters was similar. Three different alternatively used Bach2 terminal exons were identified to be located in intron 4. The inclusion of these exons resulted in the generation of Bach2 mRNA with open reading frames lacking the bZIP DNA binding domain present in the normal Bach2 protein, but retaining a partial BTB protein dimerization domain. Such Bach2 protein was excluded from the cell nucleus.ConclusionWe have identified an alternative promoter and new protein isoforms of Bach2. Our data imply that activation of an alternative promoter by proviral integration serves as a possible mechanism of up-regulation of the Bach2 gene with a potential role in B-cell lymphomagenesis. The finding of novel Bach2 transcripts and protein isoforms will facilitate a better insight into the normal and pathophysiological regulation of the Bach2 gene.

De Novo Design and Evolution of Artificial Disulfide Isomerase Enzymes Analogous to the Bacterial DsbC

The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution.

Differential Roles for the E2A Activation Domains in B Lymphocytes and Macrophages

The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A(-/-) hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages.

E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)-PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3' portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5' regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT-PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Remodeling of the Human Papillomavirus Type 11 Replication Origin into Discrete Nucleoprotein Particles and Looped Structures by the E2 Protein

The human papillomavirus (HPV) DNA replication origin ( ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.

Functional conservation of Rel binding sites in drosophilid genomes.

Evolutionary constraints on gene regulatory elements are poorly understood: Little is known about how the strength of transcription factor binding correlates with DNA sequence conservation, and whether transcription factor binding sites can evolve rapidly while retaining their function. Here we use the model of the NFKB/Rel-dependent gene regulation in divergent Drosophila species to examine the hypothesis that the functional properties of authentic transcription factor binding sites are under stronger evolutionary constraints than the genomic background. Using molecular modeling we compare tertiary structures of the Drosophila Rel family proteins Dorsal, Dif, and Relish and demonstrate that their DNA-binding and protein dimerization domains undergo distinct rates of evolution. The accumulated amino acid changes, however, are unlikely to affect DNA sequence recognition and affinity. We employ our recently developed microarray-based experimental platform and principal coordinates statistical analysis to quantitatively and systematically profile DNA binding affinities of three Drosophila Rel proteins to 10,368 variants of the NFKB recognition sequences. We then correlate the evolutionary divergence of gene regulatory regions with differences in DNA binding affinities. Genome-wide analyses reveal a significant increase in the number of conserved Rel binding sites in promoters of developmental and immune genes. Significantly, the affinity of Rel proteins to these sites was higher than to less conserved sites and was maintained by the conservation of the DNA binding site sequence (static conservation) or in some cases despite significantly diverged sequences (dynamic conservation). We discuss how two types of conservation may contribute to the stabilization and optimization of a functional gene regulatory code in evolution.

Structure of the SARS coronavirus nucleocapsid protein dimerization domain suggests a novel mechanism for helical wrapping of viral RNA

Structure of the SARS coronavirus nucleocapsid protein dimerization domain suggests a novel mechanism for helical wrapping of viral RNA

B-Myc: N-Terminal Recognition of Myc Binding Proteins

B-Myc is an endogenous, N-terminal homologue of transcription factor c-Myc that lacks the C-terminal DNA binding and protein dimerization domain of c-Myc. Clinical mutations in the c-Myc N-terminal region, and the subsequent misregulation of Myc, are implicated in the development of numerous human cancers. Myc functions to both activate and repress transcription by associating with multiple binding partners. We investigated the structural and dynamical properties of B-Myc, free or associated with the transactivation inhibitor, MM-1, and the activator, TBP, using NMR spectroscopy. B-Myc has no persistent tertiary structure, yet regions corresponding to Myc homology boxes 1 and 2 (MBI and MBII, respectively) have molten globule-like characteristics. B-Myc binds to MM-1 in a specific manner without becoming highly structured. The local regions of B-Myc involved in binding differ for MM-1 and TBP, and regions not identified by mutagenesis are found to be involved in MM-1 binding. The results provide new insights into Myc N-terminal protein-protein interactions. We propose a model for Myc regulation through differential involvement of MBI and MBII in the binding of Myc interacting proteins.

Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function.

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.

The identification and characterization of human Sister-of-Mammalian Grainyhead (SOM) expands the grainyhead-like family of developmental transcription factors.

The Drosophila gene grainyhead is the founding member of a large family of genes encoding developmental transcription factors that are highly conserved from fly to human. The family consists of two main branches, with grainyhead as the ancestral gene for one branch and the recently cloned Drosophila CP2 as the ancestral gene for the other. We now extend this family with the identification of another novel mammalian member, Sister-of-Mammalian Grainyhead (SOM), which is phylogenetically aligned with grainyhead. SOM is closely related to the other mammalian homologues of grainyhead, including Mammalian Grainyhead (MGR) and Brother-of-MGR, sharing a high degree of sequence identity with these factors in the functional DNA-binding, protein dimerization and activation domains. Protein interaction studies demonstrate that SOM can heterodimerize with MGR and Brother-of-MGR, but not with the more distant members of the family. Like grainyhead, the SOM gene too produces several distinct isoforms with differing functional properties through alternative splicing. The tissue distributions of these isoforms differ and all display highly restricted expression patterns. These findings indicate that SOM, like its family members, may play important roles in mammalian development.

The EBF/Olf/Collier family of transcription factors: regulators of differentiation in cells originating from all three embryonal germ layers.

One major mechanism for achieving cell-type-specific functions in somatic cells is the differential usage of stage- and tissue-specific genes. This is in large part regulated by transcription factors that interact directly with DNA elements controlling transcription of these genes. Several groups of transcription factors with key roles in cellular differentiation and function have been characterized, and the list of transactivating proteins with crucial functions in vivo is continuously growing. One group of evolutionarily conserved proteins with defined roles in a large variety of species are the basic helix-loop-helix (bHLH) proteins (42, 44). These factors are involved in a large number of differentiation processes, including such diverse events as sex determination in Drosophila melanogaster and B-lymphopoiesis in mice (9, 15, 38, 42, 48). In 1993, two independent reports presented a novel type of HLH protein with a dimerization domain containing two helices with homology to the second helix of the classical bHLH protein dimerization domain, but with a different type of DNA binding domain (DBD). This factor was cloned both in a Saccharomyces cerevisiae one-hybrid experiment designed to identify factors interacting with the olfactory-restricted olfactory marker protein-1 (OMP-1) promoter (69), and by biochemical purification of a factor interacting with the B-lymphocyte-restricted mb-1 promoter (27). It was accordingly named Olf-1, or early B-cell factor (EBF), which in turn led to the designation of the factor as O/E-1. Later reports showed that mice expressed at least three more members of this family, EBF2 (mMot1/O/E-3), EBF3 (O/E-2) (23, 40, 71), and O/E-4 (70), with a high degree of similarity in the DNA-binding and dimerization domains. Isolation of the O/E homologue Collier from D. melanogaster (12) provided a proof of principle for the existence of a new family of evolutionarily conserved proteins, and family members have now been cloned in several species (7, 17, 24, 27, 46, 52, 69) (Table ​(Table1).1). The biological roles of this protein family are beginning to be unraveled, and here we are compiling some of the available information about the roles of Collier/Olf/EBF (COE) proteins to give an overview of the known functions of this protein family in specific model systems and in different model species. TABLE 1. COE proteins identified to datea

Polypyrimidine-Tract-Binding Protein Affects Transcription but Not Translation of Mouse Hepatitis Virus RNA

Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5′ ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex.