Gerd G. Maul, Ph.D.

Professor
Gene Expression and Regulation Program
215-898-3817, Office

Introduction

Research in the laboratory of Gerd G. Maul, Ph.D., concentrates on how cells maintain and regulate the proper balance of a family of proteins found exclusively in the nucleus. Cells recognize DNA from foreign viruses and bacteria with help from these proteins, and so understanding how they work expands our knowledge about how cells fight off infections and other challenges.

Research Summary

Central to investigations in the Maul laboratory are structures within the nucleus - called nuclear domain 10 (ND10) - which the researchers have been determined to be holding areas or depots for regulatory proteins involved in the body's response to heavy metal exposure, hypothermia, exposure to ultraviolet light, as well as viral infection and interferon exposure. Disturbances in the proper balance of the ND10-containing proteins due to mutations in the proteins or viruses can cause malignancies such as acute promyelocitic leukemia, increased replication of viruses, and altered progression in aging. The investigators look for external triggers that prompt these sequestered regulatory proteins to be released from the depots, as well as conditions that result in recruitment into the depots. They also investigate changes in the depots themselves that affect the recruitment and release of the regulatory proteins. Results from these studies show that the cell - via the regulatory proteins stored in the ND10 depots - help the body fight acute viral infection, moderate its stress response to cadmium or short term heat exposure, segregate viral DNA and protein complexes and downregulate kinases, inflammatory proteins involved in many diseases. For example, DNA viruses such as herpes simplex virus type 1 start replicating at ND10 when they gain access to healthy cells and the cell has defense mechanisms located at ND10 that suppress infection. Recent results from the Maul laboratory suggest that cells recognize foreign complexes of DNA and protein through a mechanism that involves interaction with the ND10-associated proteins Daxx and PML. Understanding the intricate balance maintained at ND10 will help to modify that balance so that viruses are repressed and the cells defenses increased.

Recent Scientific Advances

The homeostatic balance of the cell can be disturbed by many external factors such as heat shock, heavy metal or UV light exposure, viral infection, and interferon exposure. During these externally applied insults the cell responds by various defense mechanisms such as heat shock protein upregulation, interferon production, or apoptosis (programmed cell death). Such responses need to be modulated to be appropriate to the level of the insult and such that the normal state is reached again after the cessation of the insult. Many signaling pathways have been elucidated that lead to various - often interconnected - outcomes. It has become apparent that the relative signal strength of many interactions will tilt the balance to either an activation or inactivation of certain responses or modulate the strength of these responses. One potential regulatory mechanism not investigated directly is the possibility that regulatory proteins are sequestered in specific domains in the nucleus in an inactive state and released by appropriate signals.

Earlier, researchers in the Maul laboratory described aggregations of proteins in the nucleus that had none of the traditional functions associated with the nucleus, like replication transcription, splicing, and transport (Ascoli and Maul, 1991). These nuclear domains (ND), named ND10 for their relative frequency, appeared to be involved in a number of disease processes including acute promyelocytic leukemia (Dyck et al., 1994) and viral infections (Maul, 1998) and presented the possibility that they function as depots for inactive proteins. The depot concept suggested that recruitment of regulatory proteins into and release from ND10 are regulated processes and that such changes in available proteins, brought about by regulated recruitment or release, have physiological consequences.

Although recruitment of proteins into ND10 through the interferon-induced upregulation and the SUMO-1 (small ubiquitin related modifier protein 1) modification level of PML (acute promyelocytic leukemia protein) have been defined, it is not known whether release of proteins is regulated and has physiological consequences. The researchers established that exposure to sublethal environmental stress sequentially releases ND10-associated proteins. Upon heat shock the Daxx and Sp100 proteins were released but PML, the structural protein of ND10 (Ishov et al., 1999), remained, whereas exposure to sub-toxic concentrations of CdCl2 induced the release of ND10-associated proteins including PML. In both cases, recovery times were similar and were followed by a burst of mitotic activity. Cadmium-induced release of proteins from ND10 can be blocked by inhibiting activation of the kinases p38 MAPK or ERK1/2.

On the other hand, heat-shock-induced desumofication of PML and release of proteins from ND10 are unaffected by these inhibitors, but can be recapitulated by overexpression of the SUMO-isopeptidase SENP-1. Therefore, activation of SENP-1-like SUMO-isopeptidase(s) during heat shock is not affected by these kinases. Thus, the release of ND10-associated proteins is not due to a general dispersal of nuclear domains but seems regulated by rapid desumofication during thermal stress and through the phosphorylation cascade of stress and mitogenic signaling pathways in the case of CdCl2.

Whether the release of certain proteins had consequences for ND10 depot proteins was tested for heat shock protein (Hsp) transcription and synthesis. Absence of Daxx correlated with Hsp25 induction, suggesting that Daxx normally inhibits immediate Hsp25 production. Absence of PML correlated with enhanced production of Hsp70 after CdCl2 exposure. These results suggest that segregation or release of PML or Daxx have differential physiological relevance during the stress response. The enzymatic activation of protein release from ND10 after stress resulting in variable downstream effect strengthens the concept of ND10 as a regulated depot of effector proteins (Nefkens et al. 2003 in press).

That ND10 in their function as nuclear depots have defense properties is evident in their interaction with DNA viruses (Everett and Maul, 1994; Ishov and Maul, 1996; Ishov et al., 1997). DNA viruses such as herpes simplex virus type 1 (HSV-1) start their replicative processes at ND10. The researchers have now shown that specific viral DNA sequences such as the origin of replication and viral proteins associating with this DNA are required for deposition of the infectious viral DNA at ND10. These findings appear to generalize to foreign DNA/protein complexes, since both viral and bacterial DNA/protein complexes recruit ND10 proteins, whereas cellular DNA introduced into the nucleus does not. Using the chromatin immunopreciptation assay, critical intermediates for the deposition could be demonstrated for the HSV-1 replication origin through direct or indirect binding to Daxx, and coimmunoprecipitation assay of Daxx and ICP, a viral DNA binding protein. The deposition of HSV-1 at ND10 occurs therefore most likely as a consequence of being retained at ND10 through interaction of viral genome-bound Daxx with PML of ND10. The results suggest that cells recognize specific foreign DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins Daxx and PML (Tang et al., 2003 ).

Human cytomegalovirus (HCMV) also starts its immediate-early transcription at ND10, forming a highly dynamic immediate transcript environment (ITE) at this nuclear site. The reason for this spatial correlation remains enigmatic and the mechanism for induction of transcription at ND10 unknown. The researchers investigated whether virus tegument-based transactivators are involved in the specific intranuclear location of HCMV. They demonstrated that the HCMV transactivator, tegument protein pp71, accumulates at ND10 before the production of immediate-early proteins. Intracellular trafficking of pp71 is facilitated through binding to a coiled-coil region of Daxx. The C-terminal domain of Daxx then interacts with SUMO-modified PML, resulting in the deposition of pp71 at ND10. In Daxx-deficient cells, pp71 does not accumulate at ND10 proving in vivo the necessity of Daxx for pp71 deposition. Also, HCMV form ITE at sites other than ND10 in Daxx deficient cells and so does the HCMV pp71 knockout mutant UL82-/- in normal cells. This result strongly suggests that pp71 and Daxx are essential for HCMV transcription at ND10. Lack of Daxx had the effect of reducing the productive infection rate. The researchers concluded that the tegument transactivator pp71 facilitates viral genome deposition and transcription at ND10 possibly priming HCMV for more efficient productive infection (Ishov et al., 2002).

Herpes viruses start their transcriptional cascade at ND10. The deposition of viral genomes at these nuclear sites is due to the binding of the interferon-inducible repressor proteins PML and/or Daxx to a viral DNA/protein complex. However, the presence of repressive proteins at the nuclear site of virus transcription has remained unexplained. The researchers investigated the mouse cytomegalovirus (MCMV) immediate early 1 protein (IE1), which is necessary for productive infection at low multiplicity of infections and therefore likely to be involved in overcoming cellular repression. Temporal analysis of IE1 distribution revealed its initial segregation into ND10 by binding to PML and/or Daxx and IE1-dependent recruitment of the transcriptional repressor histone deacetylase-2 (HDAC-2) to this site. However, these protein aggregates are dissociated in those cells producing sufficient IE1 through titration of PML, Daxx and HDAC-2. Importantly, binding of IE1 to HDAC-2 decreased deacetylation activity. Moreover, inhibition of HDAC by trichostatin-A resulted in an increase in viral protein synthesis, an increase in cells starting the formation of prereplication compartments and in the total infectious viruses produced. Thus, IE1, like trichostatin-A, reverses the repressive effect of HDAC evident in the presence of acetylated histones in the IE promoter region. Since HDAC also binds to the promoter region of IE1 as determined by chromatin immunoprecipitation assay, these combined results suggest that IE1 inhibits or reverses HDAC- mediated repression of the infecting viral genomes, possibly by a process akin to activation of heterochromatin. The investigators proposed that even permissive cells can repress transcription of infecting viral genomes through repressors including HDAC, Daxx and PML and the segregation of IE1 to ND10 that would inactivate those repressors. The virus can counter this repression by overexpressing IE1 when present in sufficient copy number thus reducing the availability and effectiveness of these repressors (Tang and Maul 2003).

Future research in the Maul laboratory centers on the search for additional defense actions specifically by Sp100 and its involvement in epigenetic control; Daxx, and its involvement in the cell cycle; and the innate nuclear defense against viruses based on the recognition and deposition of foreign DNA at ND10.

Selected Publications

Ascoli, C. A. and Maul, G. G. (1991). Identification of a novel nuclear domain. J Cell Biol 112, 785-795.

Dyck, J. A., Maul, G. G., Miller, W. H., Jr., Chen, J. D., Kakizuka, A. and Evans, R. M. (1994). A novel macromolecul ar structure is a target of the promyelocyte- retinoic acid receptor oncoprotein. Cell 76, 333-343.

Everett, R. D. and Maul, G. G. (1994). HSV-1 IE protein Vmw110 causes redistribution of PML. Embo J 13, 5062-5069

.Ishov, A. M. and Maul, G. G. (1996). The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition. J Cell Biol 134, 815-826.

Ishov, A. M., Stenberg, R. M. and Maul, G. G. (1997). Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment. J Cell Biol 138, 5-16.

Maul, G. G. (1998). Nuclear domain 10, the site of DNA virus transcription and replication. Bioessays 20, 660-667.

Ishov, A. M., Sotnikov, A. G., Negorev, D., Vladimirova, O. V., Neff, N., Kamitani, T., Yeh, E. T., Strauss, J. F., 3rd and Maul, G. G. (1999). PML is critical for ND10 formation and recruits the PML-interacting protein Daxx to this nuclear structure when modified by SUMO-1. J Cell Biol 147, 221-234.

Ishov, A. M., Vladimirova, O. V. and Maul, G. G. (2002). Daxx-Mediated Accumulation of Human Cytomegalovirus Tegument Protein pp71 at ND10 Facilitates Initiation of Viral Infection at These Nuclear Domains. J Virol 76, 7705-7712.

Nefkens, I., D. G. Negorev, A. M. Ishov, J. S. Michaelson, E. T. Yeh, R. M. Tanguay, W. E. Muller, and G. G. Maul. 2003. Heat shock and Cd(2+) exposure regulate PML and Daxx release from ND10 by independent mechanisms that modify the induction of heat-shock proteins 70 and 25 differently. J Cell Sci 116:513-524.

Tang, Q., and G. G. Maul. 2003. Mouse Cytomegalovirus Immediate-Early Protein 1 Binds with Host Cell Repressors To Relieve Suppressive Effects on Viral Transcription and Replication during Lytic Infection. J Virol 77:1357-67.

Tang, Q., L. Li, A.M. Ishov, V. Revol, A.L. Epstein, and G.G. Maul. 2003. Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10. J Virol. 77:5821-8.

Ishov, A.M., O.V. Vladimirova, and G.G. Maul. 2004. Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX. J Cell Sci. 117:3807-20.

Becker, K.A., L. Florin, C. Sapp, G.G. Maul, and M. Sapp. 2004. Nuclear localization but not PML protein is required for incorporation of the papillomavirus minor capsid protein L2 into virus-like particles. J Virol. 78:1121-8.

Greger, J.G., R.A. Katz, A.M. Ishov, G.G. Maul, and A.M. Skalka. 2005. The cellular protein daxx interacts with avian sarcoma virus integrase and viral DNA to repress viral transcription. J Virol. 79:4610-8.

Tang, Q., L. Li, and G.G. Maul. 2005. Mouse cytomegalovirus early M112/113 proteins control the repressive effect of IE3 on the major immediate-early promoter. J Virol. 79:257-63.

Lechner, M. S., Schultz, D. C., Negorev, D., Maul, G. G. and Rauscher , F. J. III. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain. 2005. Bio Biophy Res Com. 331:929-37.

Tang, Q. and Maul, G.G. 2005. Immediate Early Interactions and Epigenetic Defense Mechanisms - Cytomegaloviruses: Molecular Biology and Immunology; Edited by: Matthias J. Reddehase, Caister Academic Press, pp131-149.

Negorev, D., Vladimirova, O. and Maul, G.G. 2006. Interferon upregulated Sp100 isotypes selectively inhibit herpes virus transcription. J. Virol. In press

Tang, Q., Li, L., Negorev, D., and Maul, G.G. 2005. Mouse Cytomegalovirus Can Cross the Species Barrier and Replicate in Human Cells with Help from Human Cytomegalovirus Tegument Proteins or the Immediate-Early 1 protein. Submitted