Imunoglobulin G (IgG) one of the main effectors of the immune system. Contrary to antigen binding properties, which are defined by nucleotide sequence for its Fab part, effector functions of IgG are mostly defined by Fc glycosylation, which is regulated by complex interplay of dozens of genes in the IgG glycosylation pathway as well as different environmental factors. Different clones of B-lymphocytes have defined patterns of glycosylation, indicating temporal stability in the glycosylation pathway. Epigenetic regulation through DNA methylation and chromatin modifications appear the most plausible mechanism for the fixation of the particular IgG glycosylation profile in different clones. Our past and current genome wide association studies (GWAS) are identifying complex genetic network which regulates IgG glycosylation. In this project we will use several complementary experimental approaches to identify functional role of the GWAS hits in IgG glycosylation and epigenetic mechanisms responsible for the regulation of these genes. IgG glycosylation is involved in numerous diseases, including autoimmune diseases, inflammatory diseases and cancer. Several epigenetic drugs are already used to treat cancer, and many others are being developed. Understanding of the role of epigenetic regulation in IgG glycosylation will provide new targets for the development of new epigenetic drugs and new biomarkers for patient stratification.

Method of genetic and epigenetic editing, CRISPR-Cas9 system, will be used for functional validation. Epigenetic editing, i.e. modified CpG methylation, will be validated using pyrosequencing. We expect to obtain valuable results of functional relevance of our GWAS hits (BACH2, LAMB1, IL6ST, MGAT3 i IKZF1) for regulation of IgG glycosylation. Also, we expect to get results of functional validation of the gene HNF1A in regulation of fucosylation and branching of N-glycans, which have been found to be changed in diabetes of the type MODY.

Aims:

Alternative glycosylation of IgG significantly changes its function. GWA studies have identified putative gene loci associated with IgG glycosylation:

BACH2 (transcriptional factor involved in differentiation, maturation and activation of B lymphocytes)

IL6ST (signaling transporter of many cytokines)

IKZF1 (protein Ikaros, transcription factor involved in B lymphocyte differentiation)

MGAT3 (manose beta -1,4-N-acetilglucosaminiltransferase)

LAMB1 (glycoprotein of the cell matrix)

The main goal of our studies is to reveal if and how these genes are involved in establishment of the Fc glycosylation on the IgG antibodies, and to identify the mechanisms responsible for the maintenance of the specific pattern of IgG glycosylation.

Specific goals:

1. To construct CRISPR/Cas9 tools for knock-out of the BACH2, IL6ST, IKZF1, MGAT3, LAMB1 i HNF1A gene loci.
2. Functional studies of GWAS hits in hybridoma cells (mouse model) and lymphoblastoma cells (human model).
3. Analysis of N-glycans on Fc region of IgG antibodies, secreted from hybridoma cells following silencing of the targeted loci.
4. To construct epi-CRISPR tool (CRISPR/Cas9-DNMT3A) for targeted methylation of the HNF1A promoter (transcription factor and master regulator of fucosylation of N-glycans)
5. Functional validation of epi-CRISPRa for the HNF1A gene in HepG2 cells and correlation studies with s N-glycom of secreted IgG.

6. To construct CRISPR/Cas9 tools for knock-out of the BACH2, IL6ST, IKZF1, MGAT3, LAMB1 and HNF1A gene loci.