RESUMEN

Successful genome editing depends on the cleavage efficiency of programmable nucleases (PNs) such as the CRISPR-Cas system. Various methods have been developed to assess the efficiency of PNs, most of which estimate the occurrence of indels caused by PN-induced double-strand breaks. In these methods, PN genomic target sites are amplified through PCR, and the resulting PCR products are subsequently analyzed using Sanger sequencing, high-throughput sequencing, or mismatch detection assays. Among these methods, Sanger sequencing of PCR products followed by indel analysis using online web tools has gained popularity due to its user-friendly nature. This approach estimates indel frequencies by computationally analyzing sequencing trace data. However, the accuracy of these computational tools remains uncertain. In this study, we compared the performance of four web tools, TIDE, ICE, DECODR, and SeqScreener, using artificial sequencing templates with predetermined indels. Our results demonstrated that these tools were able to estimate indel frequency with acceptable accuracy when the indels were simple and contained only a few base changes. However, the estimated values became more variable among the tools when the sequencing templates contained more complex indels or knock-in sequences. Moreover, although these tools effectively estimated the net indel sizes, their capability to deconvolute indel sequences exhibited variability with certain limitations. These findings underscore the importance of judiciously selecting and using an appropriate tool with caution, depending on the type of genome editing being performed.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Mutación INDEL/genética , Genoma/genética , Genómica

RESUMEN

CRISPR-Cas9 genome editing technology has been successfully applied to generate various genetic modifications in zebrafish. The CRISPR-Cas9 system, which originally consisted of three components, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9, efficiently induces DNA double-strand breaks (DSBs) at targeted genomic loci, often resulting in frameshift-mediated target gene disruption (knockout). However, it remains difficult to perform the targeted integration of exogenous DNA fragments (knock-in) with CRISPR-Cas9. DSBs can be restored through DNA repair mechanisms, such as nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). One of our two research groups established a method for the precise MMEJ-mediated targeted integrations of exogenous genes containing homologous microhomology sequences flanking a targeted genomic locus in zebrafish. The other group recently developed a method for knocking in ~200 nt sequences encoding composite tags using long single-stranded DNA (ssDNA) donors. This chapter summarizes the CRISPR-Cas9-mediated genome modification strategy in zebrafish.

Asunto(s)

Sistemas CRISPR-Cas , Pez Cebra , Animales , Pez Cebra/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma/genética , Reparación del ADN , Reparación del ADN por Unión de Extremidades/genética

RESUMEN

Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.

Asunto(s)

Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Edición Génica , ARN Guía de Kinetoplastida , Animales , Sistemas CRISPR-Cas/genética , ARN/genética , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genética , Pez Cebra/genética

RESUMEN

Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5' and 3' junctions indicated that knock-in allele frequencies were higher at the 3' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

RESUMEN

The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (Kd), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the Kd of antigen-antibody interactions in solution. The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the Kd values of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent Kd values. This HiBiT-qIP assay showed a considerable variation in the Kd values among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.

Asunto(s)

Afinidad de Anticuerpos , Inmunoprecipitación/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Dimerización , Epítopos/química , Epítopos/inmunología

RESUMEN

To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.

Asunto(s)

Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Pollos , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo

RESUMEN

The vertebrate Zfhx1 transcription factor family comprises δEF1 and Sip1, which bind to CACCT-containing sequences and act as transcriptional repressors. It has been a longstanding question whether these transcription factors share the same regulatory functions in vivo. It has been shown that neural crest (NC) delamination depends on the Sip1 activity at the cranial level in mouse and chicken embryos, and it remained unclear how NC delamination is regulated at the trunk level. We observed that the expression of δEF1 and Sip1 overlaps in many tissues in chicken embryos, including NC cells at the trunk level. To clarify the above questions, we separately knocked down δEF1 and Sip1 or in combination in NC cells by electroporation of vectors expressing short hairpin RNAs (shRNAs) against respective mRNAs on the dorsal side of neural tubes that generate NC cells. In all cases, the migrating NC cell population was significantly reduced, paralleled by the decreased expression of δEF1 or Sip1 targeted by shRNAs. Expression of Sox10, the major transcription factor that regulates NC development, was also decreased by the shRNAs against δEF1 or Sip1. We conclude that the trunk NC delamination is regulated by both δEF1 and Sip1 in an analogous manner, and that these transcription factors can share equivalent regulatory functions in embryonic tissues.

Asunto(s)

Proteínas Aviares/metabolismo , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/embriología , Factores de Transcripción/metabolismo , Animales , Embrión de Pollo , Ratones , Cresta Neural/citología

RESUMEN

The plasmon resonant wavelength for a pair of gold nanoparticles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double-stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2-PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2-PAX6 cobinding induces a large conformation change in DC5 DNA.

Asunto(s)

ADN/química , Nanopartículas/química , Factores de Transcripción/química , Animales , Dimerización , Proteínas del Ojo/química , Oro/química , Proteínas de Homeodominio/química , Insectos , Nanopartículas del Metal/química , Conformación de Ácido Nucleico , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/química , Unión Proteica , Conformación Proteica , Proteínas Represoras/química , Factores de Transcripción SOXB1/química , Electricidad Estática , Resonancia por Plasmón de Superficie

RESUMEN

Sox transcription factors play widespread roles during development; however, their versatile funtions have a relatively simple basis: the binding of a Sox protein alone to DNA does not elicit transcriptional activation or repression, but requires binding of a partner transcription factor to an adjacent site on the DNA. Thus, the activity of a Sox protein is dependent upon the identity of its partner factor and the context of the DNA sequence to which it binds. In this Primer, we provide an mechanistic overview of how Sox family proteins function, as a paradigm for transcriptional regulation of development involving multi-transcription factor complexes, and we discuss how Sox factors can thus regulate diverse processes during development.

Asunto(s)

Diferenciación Celular , Linaje de la Célula , Factores de Transcripción SOX/metabolismo , Células Madre/citología , Animales , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , MicroARNs/metabolismo , Células Madre/metabolismo , Activación Transcripcional

RESUMEN

A binary gene expression system using the yeast GAL4 DNA-binding protein and the upstream activating sequence (UAS) of galactose-driven yeast genes is an established and powerful tool for the analysis of gene function. However, in the domesticated silkworm, Bombyx mori, this system has been limited in its utility by the relatively low transcriptional activation activity of GAL4 and by its toxicity. In this study, we investigated the potential of several established GAL4 variants (GAL4Δ, GAL4VP16, GAL4VPmad2, GAL4VPmad3, and GAL4NFκB) and of two new GAL4 variants, GAL4Rel and GAL4Relish, which contain the transcription-activating regions of the BmRel and BmRelish genes, respectively, to improve the utility of the GAL4/UAS system in B. mori. We generated constructs containing these GAL4 variants under the control of constitutive or inducible promoters and investigated their transcription-activating activity in cultured B. mori cells and embryos and in transgenic silkworms. GAL4VP16 and GAL4NFκB exhibited high transactivation activity but appeared to be toxic when used as transgenes under the control of a constitutive promoter. Similarly, GAL4VPmad2 and GAL4VPmad3 exhibited higher transactivation activity than GAL4, combined with strong toxicity. The transcription-activating activity of GAL4Δ was about twice that of GAL4. The two new GAL4 variants, GAL4Rel and GAL4Relish, were less active than GAL4. Using GAL4VP16 and GAL4NFκB constructs, we have developed a very efficient GAL4/UAS binary gene expression system for use in cultured B. mori cells and embryos and in transgenic silkworms.

Asunto(s)

Bombyx/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/genética , Plásmidos/genética , Transcripción Genética , Activación Transcripcional

RESUMEN

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.

Asunto(s)

Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Factores del Dominio POU/metabolismo , Factores de Transcripción SOX/metabolismo , Animales , Secuencia de Bases , Embrión de Pollo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores del Dominio POU/genética , Filogenia , Factores de Transcripción SOX/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Eliminación de Secuencia , Transducción de Señal , Pez Cebra

RESUMEN

The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

Asunto(s)

Tipificación del Cuerpo , Morfogénesis , Factores de Transcripción SOX/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOX/genética , Factores de Transcripción SOXB1/genética , Proteínas de Xenopus/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética

RESUMEN

Transcriptional regulatory functions of SOX proteins generally require the cooperation of partner factors that bind DNA in the vicinity of the SOX site. Each SOX-partner pair selects a specific group of regulatory target genes, with resultant gene expression patterns characterizing a particular cell differentiation state. Specific examples include the SOX2-OCT3/4 pairing in ES cells and the SOX2-PAX6 pairing in visual system primordia. When a component of a SOX-partner pair is exchanged with another factor, an overt transition of gene expression occurs in a cell, leading to the progression of developmental processes. When a SOX-partner protein pair activates its own genes, the global cell/tissue state is stabilized. Two major molecular mechanisms underlie SOX-partner factor interactions: (1) cooperative DNA binding; and (2) protein interactions dependent upon DNA binding which elicit a large transactivation potential. In vivo evidence for and molecular mechanisms of the cell specification code attributed to the SOX-partner factor complexes are reviewed.

Asunto(s)

Tipificación del Cuerpo/genética , Factores de Transcripción SOX/metabolismo , Animales , Secuencia de Bases , Desarrollo Embrionario/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Factores de Transcripción SOX/genética

RESUMEN

Many biological processes are usually coupled to the formation of protein complexes. The yeast two-hybrid system is a powerful tool for analyzing protein-protein interactions. Different patterns of protein modifications, such as glycosylation, phosphorylation, and acetylation, may affect the ability of proteins to interact. In this study, we developed the two-hybrid system that can be used in insect cells. To validate the insect two-hybrid (I2H) system, we analyzed and confirmed the known oligomer or dimer formation of silkworm Rad51 or RPA2-RPA3, respectively. The results established the feasibility of the I2H system for efficient analysis of protein interaction under conditions that closely reflect the normal physiological environment.

Asunto(s)

Bombyx/metabolismo , Proteínas de Insectos/análisis , Recombinasa Rad51/análisis , Proteína de Replicación A/análisis , Técnicas del Sistema de Dos Híbridos , Animales , Bombyx/genética , Línea Celular , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Unión Proteica , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo

RESUMEN

An enhancer trap-based GAL4-UAS system in zebrafish requires strong GAL4 activators with minimal adverse effects. However, the activity of yeast GAL4 is too low in zebrafish, while a fusion protein of the GAL4 DNA-binding domain and the VP16 activation domain is toxic to embryonic development, even when expressed at low levels. To alleviate this toxicity, we developed variant GAL4 activators by fusing either multimeric forms of the VP16 minimal activation domain or the NF-kappaB activation domain to the GAL4 DNA-binding domain. These variant GAL4 activators are sufficiently innocuous and yet highly effective transactivators in developing zebrafish. Enhancer-trap vectors containing these GAL4 activators downstream of an appropriate weak promoter were randomly inserted into the zebrafish genome using the Sleeping Beauty transposon system. By the combination of these genetic elements, we have successfully developed enhancer trap lines that activate UAS-dependent reporter genes in a tissue-specific fashion that reflects trapped enhancer activities.

Asunto(s)

Adaptación Biológica/genética , Técnicas Genéticas , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros/genética , Vectores Genéticos/genética , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Transposasas/genética , Transposasas/metabolismo , Pez Cebra/genética

RESUMEN

In higher vertebrates, the expression of Sox2, a group B1 Sox gene, is the hallmark of neural primordial cell state during the developmental processes from embryo to adult. Sox2 is regulated by the combined action of many enhancers with distinct spatio-temporal specificities. DNA sequences for these enhancers are conserved in a wide range of vertebrate species, corresponding to a majority of highly conserved non-coding sequences surrounding the Sox2 gene, corroborating the notion that the conservation of non-coding sequences mirrors their functional importance. Among the Sox2 enhancers, N-1 and N-2 are activated the earliest in embryogenesis and regulate Sox2 in posterior and anterior neural plates, respectively. These enhancers differ in their evolutionary history: the sequence and activity of enhancer N-2 is conserved in all vertebrate species, while enhancer N-1 is fully conserved only in amniotes. In teleost embryos, Sox19a/b play the major pan-neural role among the group B1 Sox paralogues, while strong Sox2 expression is limited to the anterior neural plate, reflecting the absence of posterior CNS-dedicated enhancers, including N-1. In Xenopus, neurally expressed SoxD is the orthologue of Sox19, but Sox3 appears to dominate other B1 paralogues. In amniotes, however, Sox19 has lost its group B1 Sox function and transforms into group G Sox15 (neofunctionalization), and Sox2 assumes the dominant position by gaining enhancer N-1 and other enhancers for posterior CNS. Thus, the gain and loss of specific enhancer elements during the evolutionary process reflects the change in functional assignment of particular paralogous genes, while overall regulatory functions attributed to the gene family are maintained.

Asunto(s)

Desarrollo Embrionario/genética , Evolución Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción SOXB1/genética , Homología de Secuencia de Ácido Nucleico , Animales , Secuencia de Bases , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular

RESUMEN

We analyzed molecular cascades of sex differentiation in medaka gonads by examining the transcriptional regulation of the oocyte-expressed gene, figalpha. We first confirmed that figalpha is one of the earliest marker genes of oocyte differentiation by quantitative RT-PCR and in situ hybridization. Expression of putative figalpha target genes, zpc4 and zpb, followed that of figalpha. A meiosis-specific gene, scp3, showed expression temporally and spatially similar to figalpha. To characterize the cis-regulatory sequences of figalpha, we compared genomic organizations of vertebrate figalpha genes. Both number and sequence homology of the C-terminal exons showed divergence, suggesting their less important roles. In the frog, Xenopus tropicalis, and in many teleosts, figalpha is located between hexokinase 2 and beta-adducin. We compared this genomic region for potential cis-regulatory elements and found no DNA stretches with high homology. In spite of this lack of sequence similarities, fluorescent protein transgenes surrounded with figalpha flanking sequences from the compact genomes of fugu or Tetraodon faithfully reproduced the endogenous expression of figalpha in the medaka oocytes, indicating conserved regulatory mechanisms.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Genómica , Oocitos/citología , Oocitos/metabolismo , Oryzias/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcadores Genéticos , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Despite the broad use of morpholino antisense oligonucleotides (MO) to knockdown gene function in zebrafish embryos, the efficiency of this method has not been successfully assessed, particularly in the cases of translation-blocking MOs. In our current study, we describe a luciferase assay-based system that can monitor the MO knockdown levels in zebrafish by the use of a fusion reporter construct containing the 5'-mRNA sequence of the gene of interest and the luciferase coding sequence. The decrease in luciferase activity in zebrafish embryos that have been coinjected with this reporter RNA construct and a MO that targets the gene of interest correlated well with the level of inhibition of the corresponding endogenous protein synthesis, and also with the appearance of a knockdown phenotype. This indicates the usefulness of our method. We have also found that MOs can exert considerable knockdown effects upon unintended gene targets if 15 bases or longer of contiguous homology exists between these genes and the 25-base MO in question. Our quantitative assessment method also reveals, however, that an effective and specific knockdown can be achieved when employing a strategy that takes advantage of the synergistic effect of double MOs used at low levels.

Asunto(s)

Técnicas Genéticas , Luciferasas/metabolismo , Oligonucleótidos Antisentido/química , Pez Cebra/embriología , Pez Cebra/genética , Animales , Secuencia de Bases , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Modelos Genéticos , Datos de Secuencia Molecular , Fenotipo

RESUMEN

Sox2 is universally expressed in the neural and placodal primordia in early stage embryos, and this expression depends on various phylogenetically conserved enhancers having different regional and temporal specificities. The enhancer N-3 was identified as a regulator of the Sox2 gene active in the diencephalon, optic vesicle, and after the contact of the vesicle with the ectoderm, in the lens placodal surface area, suggesting its involvement in embryonic visual system development. A 36-bp minimal essential core sequence was defined in the 568-bp-long enhancer N-3, which in a tetrameric form emulates the original enhancer activity. The core sequence comprises a SOX-binding sequence and a non-canonical PAX6 (Paired domain) binding sequence, and is activated by the synergistic action of SOX2 and PAX6 in transfected cells. The SOX and PAX6 binding sequences of the N-3 core are arranged with the same orientation and spacing as the DC5 sequence of the delta-crystallin enhancer previously demonstrated to be cooperatively bound by SOX2 and PAX6. The N-3 core sequence was also bound by these factors in a cooperative fashion, but with a higher threshold of these factors' levels than DC5, and the enhancer effect of the tetrameric sequence activated by exogenous SOX2 and PAX6 was less pronounced than that of DC5. The observations suggest that gene activation mechanisms that depend on the cooperative interaction of SOX2 and PAX6 but with different thresholds of the factor levels are crucial for the regulation of visual system development.

Asunto(s)

Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/genética , Proteínas de Homeodominio/metabolismo , Cristalino/embriología , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Proteínas de Unión al ADN/metabolismo , Inducción Embrionaria/genética , Células Epiteliales/metabolismo , Proteínas del Ojo/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/genética , Ratones , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Factores de Transcripción SOXB1 , Alineación de Secuencia , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , delta-Cristalinas/genética , delta-Cristalinas/metabolismo

RESUMEN

OBJECTIVE: Heterozygous SOX2 mutations have recently been reported to cause isolated hypogonadotropic hypogonadism (HH), in addition to ocular and brain abnormalities. Here, we report a further case with a heterozygous hypomorphic SOX2 mutation and isolated HH. PATIENT: The patient was a 28-year-old Japanese female with congenital right anophthalmia and poor pubertal development, who was found to have HH by a gonadotropin-releasing hormone test (peak serum LH, 2.3 mIU/ml; peak serum FSH, 2.9 mIU/ml). Other pituitary hormones were normal. METHODS: We performed mutation analysis of SOX2 and functional studies of mutant SOX2 protein using the core enhancer sequence of the chicken delta-1-crystallin gene (DC5) and that of the mouse nestin gene (Nes30). RESULTS: A heterozygous missense mutation (224T > A, Leu75Gln) was identified in the DNA-binding domain. The mutant SOX2 protein had a severely reduced (approximately 10%) DNA-binding affinity and a markedly diminished (20-30%) transactivation potential with no dominant negative effect. CONCLUSIONS: The results provide further support for the positive role of SOX2 in the regulation of gonadotropin production.

Asunto(s)

Proteínas HMGB/genética , Hipogonadismo/genética , Mutación Missense , Factores de Transcripción/genética , Adulto , Anoftalmos/genética , Análisis Mutacional de ADN , Femenino , Proteínas HMGB/química , Proteínas HMGB/metabolismo , Heterocigoto , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción SOXB1 , Factores de Transcripción/química , Factores de Transcripción/metabolismo