A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Alteration of Bcl11b upon stimulation of both the MAP kinase- and Gsk3-dependent signaling pathways in double-negative thymocytes.

B-cell lymphoma/leukemia 11B (Bcl11b) is a transcription factor critical for thymocyte development. We have previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double-positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double-negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt-Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and (or) Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation-associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.

Identification of a seed coat-specific promoter fragment from the Arabidopsis MUCILAGE-MODIFIED4 gene.

KEY MESSAGE: The Arabidopsis seed coat-specific promoter fragment described is an important tool for basic and applied research in Brassicaceae species. During differentiation, the epidermal cells of the Arabidopsis seed coat produce and secrete large quantities of mucilage. On hydration of mature seeds, this mucilage becomes easily accessible as it is extruded to form a tightly attached halo at the seed surface. Mucilage is composed mainly of pectin, and also contains the key cell wall components cellulose, hemicellulose, and proteins, making it a valuable model for studying numerous aspects of cell wall biology. Seed coat-specific promoters are an important tool that can be used to assess the effects of expressing biosynthetic enzymes and diverse cell wall-modifying proteins on mucilage structure and function. Additionally, they can be used for production of easily accessible recombinant proteins of commercial interest. The MUCILAGE-MODIFIED4 (MUM4) gene is expressed in a wide variety of plant tissues and is strongly up-regulated in the seed coat during mucilage synthesis, implying the presence of a seed coat-specific region in its promoter. Promoter deletion analysis facilitated isolation of a 308 base pair sequence (MUM4 0.3Pro ) that directs reporter gene expression in the seed coat cells of both Arabidopsis and Camelina sativa, and is regulated by the same transcription factor cascade as endogenous MUM4. Therefore, MUM4 0.3Pro is a promoter fragment that serves as a new tool for seed coat biology research.

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus.

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT). We found that FT expression led to very early flowering, with events showing floral buds within 1-5 months of transplanting to the glasshouse. The most rapid flowering was observed when the cauliflower mosaic virus 35S promoter was used to drive the Arabidopsis thaliana FT gene (AtFT). Early flowering was also observed with AtFT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 (PtFT1) under control of a heat-shock promoter. Early flowering trees grew robustly, but exhibited a highly branched phenotype compared to the strong apical dominance of nonflowering transgenic and control trees. AtFT-induced flowers were morphologically normal and produced viable pollen grains and viable self- and cross-pollinated seeds. Many self-seedlings inherited AtFT and flowered early. FT overexpression-induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be easily segregated away in progeny, restoring normal growth and form.

Identification and analysis of an outer-seed-coat-specific promoter from Arabidopsis thaliana.

Differentiation of the Arabidopsis thaliana (Arabidopsis) seed coat epidermal cells involves pronounced changes highlighted by the synthesis and secretion of copious amounts of dispensable, pectinaceous mucilage followed by a thick cellulosic secondary cell wall. This cell type, therefore, represents an excellent molecular-genetic model to study the biosynthesis and modification of cell wall components, particularly pectin. To support such research, we sought to identify a promoter that drives expression specifically in the Arabidopsis seed coat epidermis. Arabidopsis seed coat microarray data was analysed for genes expressed in the wild type seed coat but not the seed coat of the apetala2 mutant where the epidermal cells fail to differentiate. Of 14 candidate genes, 9 showed a seed-specific expression pattern by reverse transcriptase-PCR. Transcriptional regulatory region-ß-glucuronidase (GUS) reporter gene fusions introduced into Arabidopsis identified one promoter, that of the DIRIGENT PROTEIN1 (DP1) gene, as seed coat specific. The specificity of the expression was confirmed using a second reporter gene, Citrine YFP. Expression of both reporter genes was limited to the epidermal and palisade cell layers of the seed coat. Quantitative PCR data using wild type seed coat RNA suggested that the promoter is particularly active at 7 days post anthesis. The DP1 promoter was able to direct transcription of GUS in a similar pattern in the Brassica napus seed coat. Thus, in addition to its application in studying the plant cell wall, this promoter will provide an experimental tool for expressing high-valued recombinant proteins as well as modifying seed coat traits in economically important crops.

The Arabidopsis transcription factor LUH/MUM1 is required for extrusion of seed coat mucilage.

During differentiation, the Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells secrete mucilage composed primarily of rhamnogalacturonan I that is extruded from the seed coat upon imbibition. The mucilage of the mucilage modified1 (mum1) mutant contains rhamnogalacturonan I that is more highly branched and lacks the ability to be extruded when exposed to water. Our cloning of the MUM1 gene shows that it encodes a putative transcription factor, LEUNIG_HOMOLOG (LUH). Cellular localization and transcriptional assay results suggest that LUH/MUM1 is a nucleus-localized transcriptional activator. LUH/MUM1 is expressed in all the tissues examined, including the seed coat. Quantitative reverse transcription-polymerase chain reaction data suggest that LUH/MUM1 is expressed throughout seed coat development, reaching peak expression late in differentiation. LUH1/MUM1 expression in plants homozygous for mutations in several genes encoding regulators of seed coat mucilage was unchanged. Thus, LUH/MUM1 expression appears to be independent of other transcription factors known to regulate aspects of seed coat mucilage biology. The expression in the luh/mum1 mutant of three genes encoding enzymes needed for mucilage extrusion, MUM2, SUBSILIN PROTEASE1.7, and ß-XYLOSIDASE1, was reduced relative to that of the wild type. Overexpression of MUM2 could partially rescue the mum1 phenotype. These data suggest that LUH/MUM1 is a positive regulator of all three genes.

Hepatocyte isolation from unused/rejected livers for transplantation: initial step toward hepatocyte transplantation, the first experience from iran.

BACKGROUND: Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes can be isolated from unused/rejected livers under sterile conditions. OBJECTIVES: The quality of the hepatocytes is very important and the main and initial step in hepatocyte transplantation is hepatocyte isolation. In this study we tried to set up the methods of hepatocyte isolation in order to use the high quality cells in acute liver failure or congenital metabolic disorders. MATERIALS AND METHODS: In this study, during a year, hepatocytes were isolated from 7 unused/rejected livers among more than 300 harvested livers in Shiraz University of Medical Sciences. The two step collagenase perfusion method was used under GMP (Good manufacturing practice) for hepatocyte isolation. RESULTS: Highly quality hepatocytes with high viability and low contamination were isolated. The mean viability was 71.8% ± 21.7. In the first 4 cases microbial contamination by Staphylococci, Diphtheroid and Klebsiella was detected, however the last 3 cases were free of any micro organisms. After 5 weeks of cryopreservation in -140°C, the cell viability was still acceptable. CONCLUSIONS: Hepatocyte isolation can be performed as the main and initial step for cell transplantation from unused/rejected liver. It is the first experience in Iran.