Enzymes of the cellulose synthase (CesA) family and CesA-like (Csl) families are responsible for the synthesis of celluloses and hemicelluloses, and thus are of great interest to bioenergy research. We studied the occurrences and phylogenies of CesA/Csl families in diverse plants and algae by comprehensive data mining of 82 genomes and transcriptomes.
We found that 1) charophytic green algae (CGA) have orthologous genes in CesA, CslC and CslD families; 2) liverwort genes are found in the CesA, CslA, CslC and CslD families; 3) The fern Pteridium aquilinum not only has orthologs in these conserved families but also in the CslB, CslH and CslE families; 4) basal angiosperms, e.g. Aristolochia fimbriata, have orthologs in these families too; 5) gymnosperms have genes forming clusters ancestral to CslB/H and to CslE/J/G respectively; 6) CslG is found in switchgrass and basal angiosperms; 7) CslJ is widely present in dicots and monocots; 8) CesA subfamilies have already diversified in ferns.
We speculate that: (i) ferns and horsetails might both have CslH enzymes, responsible for the synthesis of mixed-linkage glucans and (ii) CslD and similar genes might be responsible for the synthesis of mannans in CGA. Our findings led to a more detailed model of cell wall evolution and suggested that gene loss played an important role in the evolution of Csl families. We also demonstrated the usefulness of transcriptome data in the study of plant cell wall evolution and diversity.
Cell wall; CesA; CslH; CslD; Transcriptome; Ferns; Liverworts; CGA; Gymnosperms
The cellulose synthase and cellulose synthase-like gene superfamily (CESA/CSL) is proposed to encode enzymes for cellulose and non-cellulosic matrix polysaccharide synthesis in plants. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, the global expression profiling patterns and functions of the OsCESA/CSL superfamily remain largely unknown.
A total of 45 identified members of OsCESA/CSL were classified into two clusters based on phylogeny and motif constitution. Duplication events contributed largely to the expansion of this superfamily, with Cluster I and II mainly attributed to tandem and segmental duplication, respectively. With microarray data of 33 tissue samples covering the entire life cycle of rice, fairly high OsCESA gene expression and rather variable OsCSL expression were observed. While some members from each CSL family (A1, C9, D2, E1, F6 and H1) were expressed in all tissues examined, many of OsCSL genes were expressed in specific tissues (stamen and radicles). The expression pattern of OsCESA/CSL and OsBC1L which extensively co-expressed with OsCESA/CSL can be divided into three major groups with ten subgroups, each showing a distinct co-expression in tissues representing typically distinct cell wall constitutions. In particular, OsCESA1, -3 & -8 and OsCESA4, -7 & -9 were strongly co-expressed in tissues typical of primary and secondary cell walls, suggesting that they form as a cellulose synthase complex; these results are similar to the findings in Arabidopsis. OsCESA5/OsCESA6 is likely partially redundant with OsCESA3 for OsCESA complex organization in the specific tissues (plumule and radicle). Moreover, the phylogenetic comparison in rice, Arabidopsis and other species can provide clues for the prediction of orthologous gene expression patterns.
The study characterized the CESA/CSL of rice using an integrated approach comprised of phylogeny, transcriptional profiling and co-expression analyses. These investigations revealed very useful clues on the major roles of CESA/CSL, their potentially functional complement and their associations for appropriate cell wall synthesis in higher plants.
The CELLULOSE SYNTHASE (CESA) superfamily of proteins contains several sub-families of closely related CELLULOSE SYNTHASE-LIKE (CSL) sequences. Among these, the CSLA and CSLC families are closely related to each other and are the most evolutionarily divergent from the CESA family. Significant progress has been made with the functional characterization of CSLA and CSLC genes, which have been shown to encode enzymes with 1,4-β-glycan synthase activities involved in the biosynthesis of mannan and possibly xyloglucan backbones, respectively. This review examines recent work on the CSLA and CSLC families from evolutionary, molecular, and biochemical perspectives. We pose a series of questions, whose answers likely will provide further insight about the specific functions of members of the CSLA and CSLC families and about plant polysaccharide biosynthesis is general.
CELLULOSE SYNTHASE-LIKE; mannan; xyloglucan; CSLA; CSLC; plant cell wall
An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls.
Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.
mannan; mannose; plant cell wall; glycosyltransferase; cellulose synthase like; CSL; biosynthesis; hemicellulose
Cellulose synthase-like (CSL) genes are predicted to catalyse the biosynthesis of non-cellulosic polysaccharides such as the β-d-glycan backbone of hemicelluloses and are classified into nine subfamilies (CSLA–CSLH and CSLJ). The CSLD subfamily is conserved in all land plants, and among the nine CSL subfamilies, it shows the highest sequence similarity to the cellulose synthase genes, suggesting that it plays fundamental roles in plant development. This study presents a detailed analysis of slender leaf 1 (sle1) mutants of rice that showed rolled and narrow leaf blades and a reduction in plant height. The narrow leaf blade of sle1 was caused by reduced cell proliferation beginning at the P3 primordial stage. In addition to the size reduction of organs, sle1 mutants exhibited serious developmental defects in pollen formation, anther dehiscence, stomata formation, and cell arrangement in various tissues. Map-based cloning revealed that SLE1 encodes the OsCSLD4 protein, which was identified previously from a narrow leaf and dwarf 1 mutant. In situ hybridization experiments showed that OsCSLD4 was expressed in a patchy pattern in developing organs. Double-target in situ hybridization and quantitative RT-PCR analyses revealed that SLE1 was expressed specifically during the M-phase of the cell cycle, and suggested that the cell-cycle regulation was altered in sle1 mutants. These results suggest that the OsCSLD4 protein plays a pivotal role in the M phase to regulate cell proliferation. Further study of OsCSLD4 is expected to yield new insight into the role of hemicelluloses in plant development.
CSLD; cytokinesis; leaf blade; M-phase; rice; slender leaf 1.
(1,3;1,4)-β-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-β-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-β-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-β-D-glucans in accordance with the purposes of use.
Cell wall; grasses; Hordeum vulgare; mixed-linkage glucans; polysaccharide
Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of β-mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.
In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 22.214.171.124) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.
Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.
Kaposi's sarcoma associated herpesvirus (KSHV) establishes a life-long persistent infection in B cells, which constitute the viral reservoir for reactivation and production of progeny virus. Viral reactivation is associated with multiple AIDS related malignancies including Kaposi's sarcoma, an endothelial tumor, and two B cell lymphoproliferative malignancies, the primary effusion lymphoma and the multicentric Castleman's disease. CBF1/CSL is a cellular DNA binding protein that can recruit transactivators or repressors to regulatory sites in the viral and cellular genome. The replication and transcription activator (RTA) plays an essential role in the switch between latency and lytic reactivation. RTA can either bind to DNA directly or is recruited to DNA via anchor proteins like CBF1/CSL and activates transcription. In this study we used a novel cell culture model to analyze the contribution of the CBF1/CSL protein to the process of viral reactivation in human B cells. Two isogenic CBF1/CSL proficient or deficient B cell lines were latently infected with recombinant KSHV. Lytic viral gene expression, viral replication and virus production were compared. Our results suggest that viral lytic gene expression is severely attenuated but not abolished at multiple stages before and after the onset of lytic replication while virus production is below detection levels in CBF1/CSL deficient B cells.
The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.
cellulose; cellulose synthase; CesA; CslD; CslF; comparative phylogenetics; cellulose synthase superfamily; sequence-based analysis
Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development.
(1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-β-glucan are required for brewing and distilling and high levels for positive impacts on human health.
We quantified grain (1,3;1,4)-β-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes.
We show that a region of the genome on chromosome 2 containing a cluster of CELLULOSE SYNTHASE-LIKE (Csl) genes, including CslF3, CslF4, CslF8, CslF10, CslF12 and CslH, as well as a region on chromosome 1H containing CslF9, are associated with the phenotype in this germplasm. We also observed that several regions identified by GWAS contain glycoside hydrolases that are possibly involved in (1,3;1,4)-β-glucan breakdown, together with other genes that might participate in (1,3;1,4)-β-glucan synthesis, re-modelling or regulation. This analysis provides new opportunities for understanding the genes related to the regulation of (1,3;1,4)-β-glucan content in cereal grains.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-907) contains supplementary material, which is available to authorized users.
Barley; (1,3;1,4)-β-glucan; Cell walls; GWAS; Soluble fibre
The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.
In this work, we used a comparative genomic approach to obtain new insights into the evolution of the xyloglucan-related enzymatic machinery in green plants. Detailed phylogenetic analyses were done for enzymes involved in xyloglucan synthesis (xyloglucan transglycosylase/hydrolase, α-xylosidase, β-galactosidase, β-glucosidase and α-fucosidase) and mobilization/degradation (β-(1→4)-glucan synthase, α-fucosyltransferases, β-galactosyltransferases and α-xylosyl transferase) based on 12 fully sequenced genomes and expressed sequence tags from 29 species of green plants. Evidence from Chlorophyta and Streptophyta green algae indicated that part of the Embryophyta xyloglucan-related machinery evolved in an aquatic environment, before land colonization. Streptophyte algae have at least three enzymes of the xyloglucan machinery: xyloglucan transglycosylase/hydrolase, β-(1→4)-glucan synthase from the celullose synthase-like C family and α-xylosidase that is also present in chlorophytes. Interestingly, gymnosperm sequences orthologs to xyloglucan transglycosylase/hydrolases with exclusively hydrolytic activity were also detected, suggesting that such activity must have emerged within the last common ancestor of spermatophytes. There was a positive correlation between the numbers of founder genes within each gene family and the complexity of the plant cell wall.
Our data support the idea that a primordial xyloglucan-like polymer emerged in streptophyte algae as a pre-adaptation that allowed plants to subsequently colonize terrestrial habitats. Our results also provide additional evidence that charophycean algae and land plants are sister groups.
The cellulose synthase-like gene HvCslF6, which is essential for (1,3;1,4)-β-glucan biosynthesis in barley, collocates with quantitative trait loci (QTL) for grain (1,3;1,4)-β-glucan concentration in several populations, including CDC Bold × TR251. Here, an alanine-to-threonine substitution (caused by the only non-synonymous difference between the CDC Bold and TR251 HvCslF6 alleles) was mapped to a position within HvCSLF6 that seems unlikely to affect enzyme stability or function. Consistent with this, transient expression of full-length HvCslF6 cDNAs from CDC Bold and TR251 in Nicotianabenthamiana led to accumulation of similar amounts of (1,3;1,4)-β-glucan accumulation. Monitoring of HvCslF6 transcripts throughout grain development revealed a significant difference late in grain development (more than 30 days after pollination), with TR251 [the parent with higher grain (1,3;1,4)-β-glucan] exhibiting higher transcript levels than CDC Bold. A similar difference was observed between Beka and Logan, the parents of another population in which a QTL had been mapped in the HvCslF6 region. Sequencing of a putative promoter region of HvCslF6 revealed numerous polymorphisms between CDC Bold and TR251, but none between Beka and Logan. While the results of this work indicate that naturally occurring quantitative differences in (1,3;1,4)-β-glucan accumulation may be due to cis-regulated differences in HvCslF6 expression, these could not be attributed to any specific DNA sequence polymorphism. Nevertheless, information on HvCslF6 sequence polymorphism was used to develop molecular markers that could be used in barley breeding to select for the desired [low or high (1,3;1,4)-β-glucan] allele of the QTL.
Electronic supplementary material
The online version of this article (doi:10.1007/s11032-015-0208-6) contains supplementary material, which is available to authorized users.
Barley breeding; (1,3;1,4)-β-Glucan concentration; Molecular markers; Grain quality; QTL; Transcript profiles
For Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 [HHV8]), the switch from latency to active lytic replication requires RTA, the product of open reading frame 50 (ORF50). RTA activates transcription from nearly 40 early and delayed-early viral promoters, mainly through interactions with cellular DNA binding proteins, such as CSL/RBP-Jκ, Oct-1, C/EBPα, and c-Jun. Reliance on cellular coregulators may allow KSHV to adjust its lytic program to suit different cellular contexts or interpret signals from the outside. CSL is a key component of the Notch signaling pathway and is targeted by several viruses. A search with known CSL binding sequences from cellular genes found at least 260 matches in the KSHV genome, many from regions containing known or suspected lytic promoters. Analysis of clustered sites located immediately upstream of ORF70 (thymidylate synthase), ORF19 (tegument protein), and ORF47 (glycoprotein L) uncovered RTA-responsive promoters that were validated using mRNAs isolated from KSHV-infected cells undergoing lytic reactivation. Notably, ORF19 behaves as a true late gene, indicating that RTA regulates all three phases of the lytic program. For each new promoter, the response to RTA was dependent on CSL, and 5 of the 10 candidate sites were shown to bind CSL in vitro. Analysis of individual sites highlighted the importance of a cytosine residue flanking the core CSL binding sequence. These findings broaden the role for CSL in coordinating the KSHV lytic gene expression program and help to define a signature motif for functional CSL sites within the viral genome.
The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins.
We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied.
Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.
Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL–DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain–domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL–DNA interactions and how these may impact on its transcriptional control.
Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members.
Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system.
Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family.
CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins.
We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay.
This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.
A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.
Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (Nic) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). Nic molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue ΔRAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, Nic exists exclusively in the largest Nic-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34;q34.3) translocation and constitutively express Nic. Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notchic.
Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.
Here a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) β-glucan biosynthesis gene were identified.
The oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained.
[Genbank accession number for the cloned AsPAL1 is GQ373155 and GQ379900 for AsCslF6]
PTEN is a well-described predisposition gene for Cowden syndrome (CS), a familial cancer syndrome characterized by a high risk of breast and other cancers. KLLN, which shares a bidirectional promoter with PTEN, causes cell cycle arrest and apoptosis. We previously identified germline hypermethylation of the KLLN promoter in 37% of PTEN mutation-negative CS/CS-like (CSL) patients. Patients with germline KLLN hypermethylation have an increased prevalence of breast and renal cancers when compared with PTEN mutation carriers. We have consequently sought to identify and characterize germline KLLN variants/mutations in CS/CSL and in apparently sporadic breast cancer patients. KLLN variants in CS/CSL patients are rare (1 of 136, 0.007%). Interestingly, among 438 breast cancer patients, 13 (3%) have germline KLLN variants when compared with none in 128 controls (P = 0.049). Patients with KLLN variants have a family history of breast cancer when compared with those without (P = 0.02). We demonstrate that germline KLLN variants dysregulate the cell cycle at G2. Of 24 breast carcinomas analyzed, 3 (13%) have somatic KLLN hemizygous deletions, with somatic loss of the wild-type allele in a patient with germline KLLN p.Leu119Leu. Of 452 breast carcinomas in The Cancer Genome Atlas project, 93 (21%) have KLLN hemizygous or homozygous deletions. This is the first study to associate germline KLLN variants with sporadic breast cancer and to recognize somatic KLLN deletions in breast carcinomas. Our observations suggest that KLLN may be a low penetrance susceptibility factor for apparently sporadic breast cancer.
Background and Aims
The metabolism of β-1,3 : 1,4-glucan regulates the mechanical properties of cell walls, and thereby changes the elongation growth of Poaceae plants. A previous study has shown that elongation growth of rice coleoptiles under water is enhanced by increased activity of β-1,3 : 1,4-glucan hydrolases; however, the involvement of β-1,3 : 1,4-glucan synthase activity in elongation growth under water has not yet been clarified.
The β-1,3 : 1,4-glucan synthase activity in a microsomal fraction prepared from rice seedlings grown under water was compared with that from control seedlings grown in air. The change under water in the relative expression level of CslF6, a major isoform of the β-1,3 : 1,4-glucan synthase genes, was examined by quantitative reverse-transcriptase PCR.
The level of β-1,3 : 1,4-glucan synthase activity in submerged seedlings decreased to less than 40 % of that of the control seedlings and was accompanied by a significant reduction in the amount of β-1,3 : 1,4-glucan in the cell walls. Under water, the expression of CslF6 was reduced to less than 20 % of the unsubmerged control. Bubble aeration partially restored both β-1,3 : 1,4-glucan synthase activity and the expression of CslF6 under water, correlating with suppression of the submergence-induced elongation growth of coleoptiles.
Submergence down-regulates the expression of the CslF6 gene, leading to a decreased level of β-1,3 : 1,4-glucan synthase activity. Together with the increased activity of β-1,3 : 1,4-glucan hydrolases, the decreased activity of β-1,3 : 1,4-glucan synthase contributes to the decrease in the amount of β-1,3 : 1,4-glucan in the cell walls under water. The suppression of β-1,3 : 1,4-glucan synthesis under water may be mainly due to oxygen depletion.
β-1,3 : 1,4-glucan; rice; Oryza sativa; elongation growth; cell wall; β-1,3 : 1,4-glucan synthase; CslF gene