Professor Hans Georg Zachau

Eine kurze Würdigung von Professor Hans Georg Zachau, Emeritus der LMU, mit bebilderten Annexen aus seiner aktiven Zeit sowie seiner eigenen Webseite mit Lebenslauf, Forschungstätigkeit, Publikationsliste und Übersicht zur Analyse der Immunglobulingene von Maus und Mensch

Mechanisms in Suppressing Chromosomal Translocations and Maintaining Genome Stability.

Double strand breaks (DSBs) represent one of the most dangerous forms of DNA damage. DSBs are generated during normal metabolic processes, such as DNA replication, or upon exposure of cells to exogenous agents, such as ionizing radiation. In addition, DSBs are formed as intermediates during programmed DNA rearrangements that occur during early B and T lymphocyte development, a process known as V(D)J recombination. Unrepaired or mis-repaired DNA ends can engender detrimental outcomes for cells and organisms such as aberrant genomic events like chromosomal translocations. The classical nonhomologous end joining (cNHEJ) pathway is one of the major DNA DSB repair pathways operative in mammalian cells and is required for both general DSB repair and V(D)J recombination. The studies of my dissertation investigate the functions of DNA nucleases in the repair of double strand breaks during V(D)J recombination. I have undertaken two independent, but related, lines of investigation to address this question. One project sought to elucidate the regulation of the ARTEMIS nuclease during V(D)J recombination. I examined the molecular mechanisms underlying tumorigenesis caused by an Artemis hypomorphic disease allele and identified that the ARTEMIS C-terminus suppresses tumorigenesis associated with misrepair of DNA DSBs generated during V(D)J recombination. My findings raise the possibility that particular defects in ARTEMIS that result in partial loss of function, can predispose to lymphoma, but not complete immunodeficiency. The second project focused on determining the interplay between the ARTEMIS and MRE11 nuclease in facilitating normal and aberrant V(D)J rearrangements. My results indicate that mutation of the MRE11 complex prevents tumorigenesis associated with aberrant end joining of V(D)J loci, an in turn, implicates the MRE11 complex in promoting tumorigenesis associated with DNA damage. Both projects have led to a greater understanding of the mechanisms underlying human lymphoma caused by impaired ARTEMIS nuclease activity, and additionally, identified the MRE11 complex as a possible chemotherapeutic target for improved treatment for lymphoid malignancies.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108783/1/jacheryl_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/108783/2/jacheryl_2.pd

Structural analysis of the neutralising antibody repertoire to influenza virus Haemagglutinin.

Protective immunity to influenza virus correlates with levels of neutralising antibodies directed against haemagglutinin (HA), the major membrane glycoprotein of the virus. The antibodies exert selective pressure resulting in the recurrent and annual emergence of new variant viruses. Although all five antigenic sites are implicated in antibody recognition, immunodominance is evident in the secondary antibody response, following natural infection of inbred mice as has been shown in this laboratory; BALB/c (H-2d) mAbs predominantly recognise HA1 198, and CBA/Ca (H-2k) mAbs, HA1 158, as deduced by sequencing the HA genes of X31 laboratory variants. Such restriction in the antibody response raises several questions concerning the mechanisms involved in repertoire selection and the structural basis of the selection process, which this investigation attempted to address using transgenic mice expressing human Ig μ chains.The purpose of examining the memory repertoire for influenza HA in transgenic mice was to determine whether restricted VH region gene usage and/or inability to class switch (lgM->lgG) with a resultant lack of affinity maturation would restrict the potential antibody repertoire. I have demonstrated, by structural analysis of variant virus HA genes, selected with mAbs expressing human μ chains, that there is a preferential selection for mutations within conserved residues that constitute part of the receptor binding site (HA1 225, HA1 226) that do not occur in previously documented laboratory variants. Also the majority of these variant viruses have amino acid substitutions at twp different positions: (HA1 145, 226) or (HA1 135, 225) or (HA1 135, 158) or (HA1 145, 158).In the second part of this study, I have examined the influence of concommitant immunity to X31 on re-challenge with an X31 variant virus in an attempt to mimic the human situation of recurrent infection: CBA/Ca mice were infected intranasally with X31 and their neutralising antibody repertoire investigated by hemisplenectomy, mAb production, X31 variant selection and sequencing of their HA genes. Following a rest period, mice were re-challenged with a laboratory variant, A43, differing from X31 at known antigenic sites (HA1 145, 158, del 224-230) and which was not recognised by the majority of mAbs from the primary challenge. The mAbs, established after re-infection with A43, showed heteroclitic reactivity forX31 and selected laboratory variants thereof, with substitutions at both conserved residues within the receptor binding site and at known antigenic sites (HA1 193,198, 226) or HA1 (198, 223).I have demonstrated in two different systems (a) human IgH transgenic mice, or (b) recurrent infection with a variant virus, that there is preferential selection of laboratory mutants containing multiple substitutions, including changes in conserved residues that constitute part of the receptor-binding pocket (HA1 225 or 226). This has led me to conclude that antibody affinity might play a determinant role in the selection of mutations that affect receptor binding function. The majority of X31 variants that I have cloned, and characterised, do indeed have altered receptor-binding specificity as shown by their resistance to horse serum inhibition of haemagglutination and/or altered binding to neoglycpproteins containing terminal a 2,3 or a 2,6 sialyl residues.Reduced affinity of the neutralising Ab response during infection - such as in the immunocompromised, or the very young or very old, or due to previous exposure to a related virus might skew the antibody repertoire to selection of receptor-binding variants and therefore be a further determinant of antigenic drift

The Rag2 C Terminus Participates in Repair Pathway Choice in Vivo and Suppresses Lymphomagenesis

THE RAG2 C TERMINUS PARTICIPATES IN REPAIR PATHWAY CHOICE IN VIVO AND SUPPRESSES LYMPHOMAGENESIS Vered Gigi Dr. David B Roth DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here we show that deletion of RAG2\u27s C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of lymphomas arising in a p53-/- mouse bearing a C terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites. The ability to recognize these sites is not because of specificity relaxation due to the lack of the RAG2 C terminus but probably other potential mechanisms that should be further investigated

The specificity of B-cell response in multiple sclerosis

PhDOne of the pathological features of multiple sclerosis (MS) is the presence of a long lived chronic inflammation in the central nervous system (CNS) with presence of oligoclonal IgG and IgM bands (OCBs) in the cerebrospinal fluid (CSF) derived from clonally expanded B cells. In my PhD I have tested the hypothesis that the intrathecal B cells response is antigen driven and screened putative candidate antigenic epitopes. Materials and methods: Brain tissues were supplied from The UK Multiple Sclerosis Tissue Bank. Total RNA was extracted from the brain tissues from 14 patients with MS after homogenization of the snap frozen blocks and cDNA obtained. VH and VL fragments were amplified from IgM and IgG and cloned in an in house vector to build a phage display single chain fragment variable (scFv) antibody library. The library was used to analyse the VH and VL usage, somatic mutation and clonal expansion in the MS brain and to select for scFv specific to putative autoantigens candidates. Results and discussion: Two libraries of VH only and VH plus VL gene segments from MS brain’s B cells were built. The sequences analysis has revealed a biased usage of VH and VL and evidence of clonal expansion thus supporting an antigen driven response. The auto-antigen candidates chosen for screening the libraries were the myelin basic protein (MBP)-proteolipid protein (PLP) fusion protein MP4 and specific binders were selected as highlighted with monoclonal phage ELISA. Conclusion: A MS disease specific phage display antibody library was built to facilitate the analysis of the disease specific V gene usage in the MS brain. Selection using this library has provided a proof of concept that this library is functional. The library will be used in the future to identify human antibody fragments against candidate autoantigens either for diagnostic or therapeutic applications

Doctor of Philosophy

dissertationT lymphocyte-derived malignancies are pediatric cancers often carrying poor prognoses. The proto-oncogenes underlying these malignancies frequently are also fundamental to normal lymphocyte development and function. Therefore, the discovery of heretofore unrecognized lymphocyte oncogenes and tumor suppressors is of potentially profound significance to both clinical medicine and scientific understanding. To address this, we pioneered a phenotype-driven forward-genetic screen in zebrafish (Danio rerio). Using transgenic animals with T lymphocyte-specific enhanced green fluorescent protein (EGFP), we performed chemical mutagenesis, screened fish for GFP+ tumors, and identified several lines developing heritable T cell malignancies. One of these lines, oscar the grouch (otg), is characterized by recessive inheritance. Validation of otg as a true leukemia predisposition model was accomplished using histology, immunohistochemistry, gene expression studies, confirmation of clonality, and allogeneic transplantation. In search of the genetic mutation underlying otg, we compared in vivo responses to glucocorticoids and γ-irradiation (therapies known to affect human T-ALL). Both dexamethasone (DXM) and irradiation treatment (XRT) were of limited or no utility in otg. In addition to diminished sensitivity, otg larvae were resistant to radiation-induced apoptosis and showed decreased activation of caspase 3. We determined this resistance is due to a block in the intrinsic mitochondrial apoptosis pathway. To discover genetic changes that may cause T-ALL, and those contributing to relapse, we used array comparative genomic hybridization (aCGH) in both humans and zebrafish. We identified copy number aberrations (CNAs) in 17 zebrafish T-ALLs, and compared all D. rerio genes found in any CNA to a cohort of 75 human T-ALL samples. We found significant overlap (62%) with genes in CNAs from the human T-ALLs. Additionally, 15 genes recurrently altered (≥3 samples) in zebrafish T-ALL were also in CNAs from 5 or more human T-ALLs. Finally, we used aCGH to study CNA genes acquired during iterative allo-transplantations of 3 zebrafish malignancies. We compared these genes with human aCGH results from 23 patients who either failed induction, or had already relapsed. Again, we observed a large overlap (53%) as well as 9 genes found in 2 fish and ≥5 humans. These genes are candidates for causing the increased severity of these tumors

Control of lineage commitment in acute leukaemia

PhD ThesisAcute leukaemia with the t(4;11) translocation is strongly associated with pro B-acute lymphoblastic phenotype. Here is described a lineage switch from acute lymphoblastic leukaemia (ALL) to acute myeloid leukaemia (AML) which carries identical t(4;11) breakpoints that provides insight into regulation of lineage commitment and the haematopoietic origin of leukaemia. Stable DNA microsatellite sequences argue against a therapy-related AML. Genome sequencing and RNAseq identified 12 novel and deleterious mutations unique to the AML. Immunoglobulin rearrangement analysis suggested the common cell of origin lied within a population prior to B cell differentiation. Sorting of haematopoietic stem/progenitor cell populations followed by multiplex PCR and next generation sequencing for the fusion and secondary mutations demonstrated the occurrence of the leukaemogenic MLL-AF4 fusion gene in cell populations as early as the multipotent progenitor, MPP, population in both ALL and AML. In this most primitive population, the AML carries mutations in chromatin modulating genes CHD4 and PHF3, suggesting their importance in lineage commitment. Knockdown CHD4 and PHF3 individually and in combination in the pro-B ALL t(4;11) SEM cell line resulted in ~3 fold higher expression of the myeloid cell surface marker CD33. Further analysis was performed using a recently described model of MLL-AF4 leukaemogenesis consisting of CD34+ cord blood cells transduced with a chimeric MLL-Af4 fusion gene. Knockdown of CHD4 and PHF3 resulted in loss of lymphoid differentiation potential in vitro. Analysis of different PHF3 splice variants revealed that only mutation-carrying PHF3 variants increased CD33 on SEM cells and that a balance between PHF3 variants was required for the lineage fidelity. This study suggests that the ALL and AML share a common primitive cell of origin and that mutations in CHD4 and PHF3 shift the lymphoid phenotype towards a myeloid lineage leukaemia

An investigation of the molecular basis of interactions between human monoclonal antibodies and antigens that are clinically relevant in systemic lupus erythematosus and the Antiphospholipid Syndrome.

Autontibodies to a wide variety of antigens are associated with systemic lupus erythematosus (SLE) and the Antiphospholipid Syndrome (APS). Previous studies have demonstrated the importance of somatic mutations and arginine residues in the complementarity determining regions (CDRs) of pathogenic anti-dsDNA and antiphospholipid antibodies. This thesis describes the study of two human monoclonal IgG antibodies, B3 (anti-DNA) and IS4 (antiphospholipid) that were derived from a patient with active SLE and primary APS respectively. I have demonstrated in-vitro expression and mutagenesis of B3 and IS4 and used this expression system to investigate the importance of the arginine residues in B3VH and IS4VH. The mutant heavy chains, as well as the wild-type VH were expressed with different light chains and the resulting antibodies assessed for binding to nucleosomes, alpha-actinin, cardiolipin (CL), phosphatidylserine (PS), beta-2-glycoprotein I (foGPI), and the N-terminal domain of p2GPI (Domain I) using direct binding assays. The results obtained have shown that the presence of arginine at position 53 in B3VH was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of this arginine reduced binding to alpha-actinin, pzGPI and Domain I of P2GPI. The fact that the arginine to serine substitution at position 53 in B3VH significantly alters binding of B3 to different clinically relevant antigens, but in opposite directions implies that this arginine residue plays a critical role in the affinity maturation of the antibody B3. Furthermore, of four arginine residues in IS4VH CDR3 substituted to serine, two at positions 100 and 100g reduced binding to all antigens, while two at positions 96 and 97 reduced binding to fcGPI but increased or decreased binding to CL and PS. Only one H/L chain combination bound neutral phospholipid and none bound dsDNA hence, these effects are particularly relevant to antigens important in APS. Therefore, my findings suggest that these four arginine residues have developed as a result of somatic mutations driven by an antigen containing both phospholipid and frGPI. These results extend our knowledge of the structure-function relationship of human anti-DNA and anti phospholipid antibodies and aid in our understanding of how these antibodies lead to pathogenicity and what we need to target in the future for possible therapies