stor@uchicago.edu

Universitaet Freiburg, Germany, M.D.

Research Summary

Gene expression is controlled by activation and repression. Repression can be caused by methylation of cytosine in the sequence 5'CpG3'. 70% of CGs are methylated in mammals. Preventing DNA methylation is embryonic lethal because it results in uncontrolled gene activation. Very little is known about how methylation is targeted. The methylation modifier gene, Ssm1, discovered by our laboratory, is a candidate for encoding such a novel targeting function. When a transgene, HRD, comes under the influence of Ssm1, it is highly methylated at CGs and not expressed. Ssm1 acts early in embryonic development. It seems to direct methyl-transferases to its target genes. Only after DNA methylation does the target gene adopt an inactive chromatin state and cease to be transcribed. We have mapped Ssm1 to a small interval in the mouse genome and have used positional cloning to identify the Ssm1 gene. Ssm1 encodes a KRAB-ZincFinger protein. We postulate that the Zinc fingers bind DNA sequences the embryo needs to inactivate and that the KRAB domain interacts with other proteins that cause methylation of the marked DNA sequences. The characterization of Ssm1 and the determination of its endogenous targets and effects throughout development are of major importance. It will help to understand how genes are targeted for silencing in normal development and cancer.

Another project is the somatic hypermutation (SHM) of immunoglobulin genes that encode antibodies for immunity. Antibodies are produced by B lymphocytes. When these cells encounter a foreign substance, such as bacteria or viruses, they undergo a very high rate of SHM of the expressed antibody genes. SHM is initiated by a cytidine deaminase changing cytosines into uracils. In other genes, such uracils are repaired by base excision repair. However, in antibody genes during SHM, error-prone DNA polymerases introduce more errors into all four bases (A, C, G, T). In this fashion, the affinity of the antibodies vastly increases, aiding the destruction of infectious agents or cancer cells. The molecular details of the mutation mechanism, including transcription, error prone DNA repair, and the role of chromatin are a major focus of our laboratory.

Under the control of the modifier Ssm1, the HRD transgene undergoes strain-specific DNA methylation. Bisulfite analysis shows that CpG dinucleotides are almost completely methylated (red marks) in every transgene sequence in adult B6 (black mouse) but very little in D2 (beige mouse). Mice of the D2 strain express the transgene throughout life. B6 early embryos express the transgene, but starting around day 6 of embryogenesis, B6 mice cease expression when CpGs are completely methylated and inactivating chromatin changes have taken place.

Selected Publications

Kodgire, P., Mukkawar, P., North, J.A., Poirier, M. G., and Storb, U. (2012) Nucleosome stability dramatically impacts the targeting of somatic hypermutation. Mol. Cell Biol., 32:2030-2040. (PubMed)

Ratnam, S., Bozek, G., Nicolae, D., and Storb, U. (2010). The pattern of somatic hypermutation of Ig genes is altered when p53 is inactivated. Mol. Immunol., 47:2611-2618. (PubMed)

Tanaka, A., Shen, H., Ratnam, S., and Storb, U. (2010). Attracting AID to targets of somatic hypermutation. J. Exp. Med., 207:405-415. (PubMed)

Storb, U., Shen, H., and Nicolae, D. (2009). Somatic hypermutation: Processivity of the cytosine deaminase AID and error-free repair of the resulting uracils. Cell Cycle, 8:3097-3101. (PubMed)

Shen, H.M., Poirier, M.G., Allen, M., North, J., Lal, R., Widom, J., and Storb, U. (2009). The activation induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes, but only during transcription. J.Exp Med., 206:1057-71. (PubMed)

Longerich, S., Orelli, B., Martin, R., Bishop, D., and Storb, U. (2008). Brca1 in Ig gene conversion and somatic hypermutation. DNA Repair, 7: 253-266. (PubMed)

Longerich, S., Meira, S., Shaw, D. Samson, L., and Storb, U. (2007). Alkyladenine glycosylase (Aag) in somatic hypermutation and class switch recombination. DNA Repair, 6:1764-1773. (PubMed)

Shen H., Tanaka, A., Bozek, G., Nicolae, D., and Storb, U. (2006). Somatic hypermutation and class switch recombination in Msh6-/-Ung-/- double-knockout mice. J. Immunol., 177:5386-5392. (PubMed)

Shen, H., Ratnam, S., and Storb, U. (2005). Targeting of the activation-induced cytosine deaminase (AID) is strongly influenced by the sequence and structure of the targeted DNA. Mol. Cell. Biol., 25:10815-10821. (PubMed)

Longerich, S., Tanaka, A., Bozek, G., Nicolae, D. and Storb, U. (2005). The very 5' end and the constant region of Ig genes are spared from somatic mutation because AID does not access these regions. J. Exp. Med., 202:1443-1454. (PubMed)

Padjen, K., Ratnam, S., and Storb, U. (2005). DNA methylation precedes chromatin modifications under the influence of the strain-specfici modifier Ssm1. Mol. Cell. Biol., 25: 4782-4791. (PubMed)

Shen, H. M. and Storb, U. (2004). Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled. Proc. Natl. Acad. Sci. U S A, 101: 12997-13002. (PubMed)

Michael, N., Shen, H. M., Longerich, S., Kim, N., Longacre, A. and Storb, U. (2003). The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription. Immunity, 19: 235-42. (PubMed)

Sun, T., Clark, M. R. and Storb, U. (2002). A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling. Immunity, 16: 245-55. (PubMed)

Michael, N., Martin, T. E., Nicolae, D., Kim, N., Padjen, K., Zhan, P., Nguyen, H., Pinkert, C. and Storb, U. (2002). Effects of sequence and structure on the hypermutability of immunoglobulin genes. Immunity, 16: 123-34. (PubMed)

bernard.roizman@bsd.uchicago.edu

Sc.D., Johns Hopkins, 1956

Research Summary

Herpes simplex viruses cause two kinds of infections. On initial infection at the portal of entry into the body, the virus replicates and kills the infected cell (lytic infection). In the course of its replication, the virus infects nerve endings and is transported retrograde to neuronal nuclei of peripheral ganglia where it remains latent (silent) and does not harm the neuron. In some individuals the virus periodically reactivates, and is transported anterograde to a site near the portal of entry where it can cause recurrent lesions.

The focus of the research conducted in this laboratory is on the mechanism by which herpes simplex virus with less than 100 genes takes over a human cell with more than 20,000 genes in both lytic and latent infection.

Current studies on lytic infection indicate that both the virus and the cell have evolved a large number of functions designed to thwart each other’s efforts. The cell attempts to silence viral DNA and preclude the expression of its genes. The virus has evolved a large number of functional domains in its proteins that are designed to silence the cell and prevent it from shutting down the virus. In the course of these studies we have identified both the mechanisms by which the cell attempts to shut down the virus and virus-encoded functions that instead silence the cell and block the activation cellular innate immune responses.

The exciting results of the studies on latent infection are two fold: in peripheral neurons the virus allows itself to be silenced by host repressors.  At the same time it has evolved the machinery that enables it to reactivate when the neuron harboring the latent virus is stressed by external stimuli such as fever, UV light or emotional/hormonal stress.

The laboratory has trained approximately 50 graduate students and approximately the same number of post-doctoral fellows. The majority of the trainees are in Universities engaged in research and teaching.

Selected Publications

Kalamvoki M, Du T, Roizman B. Cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing STING, viral mRNAs, and microRNAs. Proc Natl Acad Sci U S A. 2014 Nov 18;111(46):E4991-6. (PubMed)

Kalamvoki M, Roizman B. HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation. Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):E611-7. (PubMed)

Du T, Zhou G, Roizman B. Modulation of reactivation of latent herpes simplex virus 1 in ganglionic organ cultures by p300/CBP and STAT3. Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):E2621-8. (PubMed)

Zhou G, Du T, Roizman B. The role of the CoREST/REST repressor complex in herpes simplex virus 1 productive infection and in latency. Viruses. 2013 Apr 29;5(5):1208-18. Review. (PubMed)

Du T, Zhou G, Roizman B. HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18820-4. (PubMed)