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BPM1 interacts with components of the DDR complex and stimulates DNA methylation at CHH sites, suggesting its involvement in the RdDM methylation pathway. The best-known function of MATH-BTB proteins, including Arabidopsis BPM proteins, is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin-proteasome pathway. This paper reports a new CUL3-independent role of BPM1 in RNA-directed DNA methylation (RdDM). Using quantitative and qualitative Y2H, pull down, microscale thermophoresis and FRET-FLIM, we demonstrate that BPM1 interacts with DMS3 and RDM1, components of the chromatin remodeling DDR complex involved in the recruitment of the RdDM methylation machinery. All three proteins colocalized predominantly in the nucleus. The MATH domain, which specifically binds proteins destined for degradation, was not essential for interactions with DMS3 and RDM1. In plants overexpressing BPM1, endogenous DMS3 protein levels were stable, indicating that BPM1 does not induce proteasomal degradation. In RDM1-overexpressing plants, RDM1 was not ubiquitinated. Together, these results suggest that BPM1 does not mediate the degradation of DMS3 and RDM1. Additionally, overexpression of BPM1 caused increased global methylation levels as well as CHH methylation in promoters of two RdDM-regulated genes, FWA and CML41. Overall, BPM1 seems to have a stimulating effect on RdDM activity, and this role appears to be unrelated to its known function as a Cul3-based E3 ligase adaptor.

Glycans are an essential structural component of Immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation). We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in thein vitroIgG expression system. Up- and downregulation of three genes,EEF1A1, MANBAandTNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in thisin vitroexpression system.

Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.

Glycosylation of immunoglobulin G (IgG) largely defines function of this antibody and regulates inflammation on multiple levels but the mechanisms and the genes involved in the process of IgG glycosylation are still vaguely understood. Genome wide association studies (GWAS) enabled identification of many genes potentially involved in IgG glycosylation but in the most cases functional validation is missing. Composition of the IgG glycome changes significantly with various diseases and ageing. In women, the most prominent change occurs during the perimenopausal period and a recent intervention study revealed that estrogen regulates IgG glycosylation. The most affected IgG glycome features are galactosylation, sialylation and structures with bisecting GlcNac. Analysis of the Signaling Pathway Projects (SPP) web knowledgebase revealed that E2 affects expression of four genes with yet unknown role in IgG glycosylation with three of them associated with galactosylation (RUNX1, RUNX3 and SPINK4) and one associated with sialylation (ELL2) through previous GWA studies. To map downstream signaling mechanisms responsible for the effect of estrogen on IgG glycome composition we utilized our recently developed in vitro IgG expression system HEK293-FS for targeted CRISPRa/CRISPRi manipulation of candidate loci. This system exploits stably integrated dCas9-VPR or dCas9-KRAB expression cassettes for targeted upregulation or downregulation of genes via transient transfection of FreeStyleTM 293-F cells with specific gRNAs. We successfully upregulated or downregulated RUNX1, RUNX3, SPINK4 and ELL2 but only upregulation of RUNX3 (Runt-related factor 3) and SPINK4 (Serine Peptidase Inhibitor Kazal Type 4) resulted with concomitant change in IgG glycome composition. Upregulation of RUNX3 resulted with significant decrease in galactosylated glycans accompanied with increase in agalactosylated glycans. Upregulation of SPINK4 was also accompanied with decrease in galactosylated glycans but the ratio of agalactosylated glycans remained stable. To further explore the role of SPINK4 and RUNX3 in IgG galactosylation we measured B4GALT1 (beta-1, 4- galactosyltransferase 1) expression after upregulation and observed no significant change in B4GALT1 transcript levels. This suggests that RUNX3 and SPINK4 do not suppress B4GALT1 on transcription level, but through some indirect pathway that should be further investigated. Taken together, the results of this study suggest the molecular mechanism through which E2 could regulate IgG glycosylation, specifically galactosylation.

Pro- or anti-inflammatory activity of immunoglobulin G (IgG) depends largely on differential N- glycosylation of its Fc region. Alternative glycosylation affects binding efficiency of this antibody to antigens, ligands, or different complexes of the immune system, which modulates immune response. While previous genome-wide association studies (GWAS) succeeded to associate functional network of gene loci with IgG glycosylation pleiotropic with inflammatory diseases, it is still unclear whether these genes have a functional importance and if so, how they are involved in this process. For this purpose, we applied already established system based on FreeStyle™ 293-F cells, with stably integrated CRISPR/dCas9 fusions (dCas9-VPR or dCas9-KRAB) for direct regulation of the candidate genes using appropriate sgRNAs. The cell system is designed to secrete IgG molecules, so that glycans can be analyzed as the final phenotype following gene manipulations. The GWA hits were grouped with respect to different glycosylation traits such as galactosylation (KIF3C, NFKB1, MANBA, SLC38A10, TNFRSF13B, EEF1A1, HIVEP2), fucosylation (MYCBP, ACVR1C, KIF11, TBX21, TBKBP1, NFATC2), sialylation (IL6ST, GGA2, SLC17A9, SPPL3) and bisecting GlcNAC (TCF3, SMARCB1, DERL2, RRBP1, KDELR2). Out of seven genes associated with galactysylation only MANBA, NFKB1, TNFRSF13B and EEF1A1 showed change of agalactosylated structures when upregulated using dCas9-VPR. Out of six hits associated with fucosylation, only upregulation of TBX21 and TBKBP1 showed significant changes in galactosylation, but we did not detect any changes in fucosylation. This can suggest that a different cell model might be preferable to validate if these GWA hits are responsible for fucosylation, since glycans on IgG produced from FreeStyle™ 293-F cells possess only core fucose. Out of five loci associated with bisecting GlcNac, only the upregulation of KDELR2 produced an increase in biantennary glycan structures with bisecting GlcNac. The upregulation of DERL2 and RRBP1 resulted in an increase of digalactosylated biantennary glycans. Out of four GWA hits for sialylation, only downregulation of SPPL3 led to hyperglycoslation with the corresponding increase in sialylated and galactosylated structures and decrease in agalactosylated glycans, even though all the genes were successfully up - or downregulated. In conclusion, in our cell model, which produces human plasma-like IgG, we have proven the functional role of several GWA hits which are not glycosyltransferases but are associated with the IgG glycosylation pathway. Ongoing research will unravel the exact role of these genes in an integrated picture of molecular mechanism regulating IgG glycosylation.

Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. One of the best examples is a simple fucosylation that can switch IgG effector function between pro- and anti- inflammatory, which is critical in production of therapeutic antibodies. Several genome-wide association studies revealed a network of genes associated with IgG glycosylation which are pleiotropic for several diseases. To understand the complex molecular mechanisms that regulate IgG glycosylation, we designed a versatile system for IgG production and gene manipulation that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR/dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells, essentially creating derived cell lines with integrated machinery for programmed transcriptional control. Transient transfection of those cells with the second component of the system, a plasmid for monocistronic expression of both IgG chains and co-expression of gRNA molecules, enables the readout of IgG glycan profiles following precisely targeted up- or downregulation of genes of interest. Though the development of the system was complex, its utilization by the end user is simple and straightforward. Transfection of the large dCas9 fusion constructs is quite inefficient and represents a bottleneck in effective experimental design with high throughput. The bipartite nature of the system ensures that only the cells expressing gRNA (with concomitant transcriptional alteration) produce IgG that is analyzed for glycosylation profile. We validated our derived cell lines that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glycogenes with a known role in IgG glycosylation. Transcriptional up- and downregulation of B4GALT1, encoding the enzyme that adds galactose to the core glycan structure, had the predictable effect on the abundance of agalactosylated and galactosylated structures, changing the glycosylation profile depending on the transcript level. Similarly, downregulation of FUT8, encoding a fucosyltransferase, decreased the core-fucosylated structures, while upregulation of ST6GAL1 and MGAT3 increased sialylated structures and those with a bisecting GlcNAc, respectively, which was in line with the expected change in enzyme levels. Interestingly, the change in sialylated structures was accompanied by changes in galactosylation, again demonstrating the complexity of the IgG glycosylation network. Our newly developed system for transient expression of can be used for studying the role of genes associated with IgG glycosylation by GWA studies. It can also be easily repurposed to serve as a model for other proteins and their posttranslational modifications, with appropriate targeting via gRNA.

Članovi proteinske porodice MATH-BTB sudjeluju u ubikvitinskom putu degradacije proteina kao adaptori E3 ligaze ovisne o kulinu 3 i odgovorni su za specifičnost prema ciljanom supstratu. U uročnjaku (Arabidopsis thaliana L.) postoji šest gena ove obitelji (BPM 1-6). Protein BPM1 uz domene MATH i BTB sadrži i konzerviranu domenu SPOP koja zajedno s domenom BTB sudjeluje u stvaranju proteinskih interakcija s kulinom 3. Domena MATH sudjeluje u selektivnim interakcijama s proteinima ciljanim za proteasomsku razgradnju. Takve interakcije utvrđene su između proteina BPM1 i nekih transkripcijskih faktora poput HB6 koji ima ulogu u odgovorima reguliranim apscizinskom kiselinom u biljnim stanicama. Preliminarna istraživanja su pokazala interakciju proteina BPM1 s proteinima DMS3 i RDM1 koji sudjeluju u metilaciji DNA posredovanoj molekulama RNA. U ovom radu interakcije proteinskih domena BPM1 s transkripcijskim faktorom HB6 i proteinima DMS3 odnosno RDM1 su analizirane metodom kopurifikacije „jedan na jedan“. Konstruirani su odgovarajući ekspresijski plazmidi, optimizirani su postupci poticanja ekspresije i pročišćavanja za svaki pojedini protein ili varijantu proteina te su eksprimirani i pročišćeni rekombinantni proteini iz kemijski transformiranih bakterijskih stanica. Interakcija proteina je provjerena hibridizacijom po Westernu i metodom SDS-PAGE. Potvrđena je interakcija proteina BPM1 i DMS3 posredovana domenom MATH te interakcija BPM1 s RDM1 koja nije specifično posredovana niti jednom pojedinom domenom. Interakcija proteina BPM1 i HB6 nije dokazana uslijed degradacije proteina HB6 . Members of MATH-BTB protein family partake as adaptors of cullin 3 dependant E3 ligase in the ubiquitin proteasome pathway and are responsible for specificity towards a substrate. In Arabidopsis thaliana there are 6 genes from this family (BPM 1-6). BPM1 protein along MATH and BTB domains, contains conserved SPOP domain which together with BTB domain partakes in forming of protein interactions with cullin 3. MATH domain has a role in selective interactions with proteins which are targeted for proteasomal degradation. This kind of interactions are established between BPM1 protein and some transcriptional factors like HB6 which has an important role in abscisic acid dependant response in plant cells. Preliminary research have shown an interaction between BPM1 protein and DMS3 and RDM1 proteins which partake in a DNA methylation directed by RNA molecules. Here interactions between BPM1 protein, HB6 transcription factor and proteins DMS3 and RDM1 were analized with „one to one“ pull down method. Adequate plasmid constructs were made, conditions for induced expression and purification for each protein and protein variant were optimised and recombinant proteins were expressed and purified from chemically transformed bacterial strains. Protein-protein interactions were proven by Western blot and SDS-PAGE. Interaction between BPM1 and DMS3 was confirmed and it is primarily mediated by MATH domain. Positive interaction between BPM1 and RDM1 didn't show preference towards any BPM1 domain. BPM1 and HB6 interaction wasn't confirmed due to HB6 degradation.