BACKGROUND: Cornelia de Lange syndrome (CdLS) is mainly characterized by specific facial features, growth retardation, and bone deformities. Seven genes reportedly cause CdLS. Recent research has reported that loss-of-function variants affecting MAU2, which encodes a regulator of the cohesin complex, can cause CdLS. Thus far, only one MAU2-CdLS case has been reported worldwide.
METHODS: We detected a novel variant in MAU2 gene, NM_015329, c.526C>T (p.Arg176Trp) in a Chinese patient with CdLS, constructed a plasmid for in vitro transcriptional and protein level analysis, and analyzed the interaction between the MAU2/NIPBL complex using molecular dynamics (MD).
RESULTS: The results showed that the level of the exogenous MAU2 mutant protein was significantly reduced compared with that of the exogenous wild-type protein. However, MD analysis predicted an increased binding free energy between the MAU2 and NIPBL proteins that may impact the structural stability of the complex.
CONCLUSIONS: We investigated a MAU2-CdLS case in a Chinese family, which strengthens the association between MAU2 variants and CdLS phenotypes. We therefore propose that MAU2 be included in the CdLS gene screening list.

Introduction: β-Alanine is the only β-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of β-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method. Methods: In this study, we constructed an Escherichia coli recombinant strain for efficient β-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome. Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of β-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the β-alanine content. The key cellulosome elements, dockerin (docA) and cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. β-alanine production reached 7.439 mg/L and 25.87 mg/L in the two engineered strains. The β-alanine content reached 755.465 mg/L in a 5 L fermenter. Discussion: The content of β-alanine synthesized by constructed β-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of β-alanine using a cellulosome multi-enzyme self-assembly system.

Structural maintenance of chromosomes (SMC) proteins are critical to maintain mitotic fidelity in all organisms. Over the last decades, acute inactivation of these complexes, together with the analysis of their dynamic binding to mitotic chromatin, has provided important insights on the molecular mechanism of these complexes as well as into the consequences of their failure at different stages of mitosis.Here, we describe a methodology to study both SMC function and dynamics using Drosophila melanogaster syncytial embryos. This system presents several advantages over canonical inactivation or imaging approaches. Efficient and fast inactivation of SMC complexes can be achieved by the use of tobacco etch virus (TEV) protease in vivo to cleave engineered versions of the SMC complexes. In contrast to genetically encoded TEV protease expression, Drosophila embryos enable prompt delivery of the protease by microinjection techniques, as detailed here, thereby allowing inactivation of the complexes within few minutes. Such an acute inactivation approach, when coupled with real-time imaging, allows for the analysis of the immediate consequences upon protein inactivation. As described here, this system also presents unique advantages to follow the kinetics of the loading of SMC complexes onto mitotic chromatin. We describe the use of Drosophila embryos to study localization and turnover of these molecules through live imaging and fluorescence recovery after photobleaching (FRAP) approaches.

The cohesin protein complex regulates multiple cellular events including sister chromatid cohesion and gene expression. Several distinct human diseases called cohesinopathies have been associated with genetic mutations in cohesin subunit genes or genes encoding regulators of cohesin function. Studies in different model systems, from yeast to mouse have provided insights into the molecular mechanisms of action of cohesin/cohesin regulators and their implications in the pathogenesis of cohesinopathies. The zebrafish has unique advantages for embryonic analyses and quantitative gene knockdown with morpholinos during the first few days of development, in contrast to knockouts of cohesin regulators in flies or mammals, which are either lethal as homozygotes or dramatically compensated for in heterozygotes. This has been particularly informative for Rad21, where a role in gene expression was first shown in zebrafish, and Nipbl, where the fish work revealed tissue-specific functions in heart, gut, and limbs, and long-range enhancer-promoter interactions that control Hox gene expression in vivo. Here we discuss the utility of the zebrafish in studying the developmental and pathogenic roles of cohesin.

Silenced heterochromatin influences all nuclear processes including chromosome structure, nuclear organization, transcription, replication, and repair. Proteins that mediate silencing affect all of these nuclear processes. Similarly proteins involved in replication, repair, and chromosome structure play a role in the formation and maintenance of silenced heterochromatin. In this chapter we describe a handful of simple tools and methods that can be used to study the atypical role of proteins in gene silencing.