Dr. Aladjem's photoMirit I. Aladjem, Ph.D.

Senior Investigator, Laboratory of Molecular Pharmacology,

DNA Replication Group

email Mirit Aladjem
 

Dr. Aladjem received her Ph.D. from Tel Aviv University.  She was a research associate at the Weizmann Institute of Science and then a postdoctoral fellow at the Salk Institute. In the course of her postdoctoral research, Dr. Aladjem was awarded an EMBO postdoctoral fellowship, a human Frontiers postdoctoral fellowship, and was a special fellow for the Leukemia Society of America.  Working with Dr. Geoffrey M. Wahl, her studies at the Salk focused on the initiation of DNA replication and cell cycle control in mammalian cells.   Dr. Aladjem joined the Laboratory of Molecular Pharmacology in October, 1999.


Dr. Aladjem is organizing the Cell Cycle Interest Group Symposium on the 14th day of May,  2007.  Details are available here.
 

Research Interests


(Click on the numbers for links to more detailed information and pertinent publications.  A complete publication list for Dr. Aladjem is available here)

Research at the DNA replication group aims at understanding how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on an intricate network of signaling molecules that monitor cellular metabolism and environmental signals. This network insures that cells do not start duplicating their genetic material under unfavorable conditions, such as the absence of sufficient nutrients or the presence of potentially damaging agents. When DNA replication escapes cell cycle controls, the results are developmental abnormalities, genomic instability, and cancer.

Knowledge about molecules that regulate cell growth has increased exponentially in recent years, but our ability to make sense of this detailed information has not. Moreover, although chromatin is a major target for cell cycle signaling, very little is known about how cells respond to these signals at the chromatin level.   Our studies probe into this missing link through the analysis of molecular factors that determine the site and the timing of DNA replication.  In parallel, we are engaged in a collaborative effort to depict cellular signaling networks that control the mammalian cell cycle.

To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. We have used the human beta globin locus as a model to delineate the sequence requirements for dictating the location of starting points for  DNA replication (Aladjem, 1998; Wang, 2004; Wang, 2006).  We had also reported that the timing of DNA replication during the S-phase of the cell cycle can be altered (Lin, 2003).  We are now elucidating the genetic and epigenetic factors that determine replication timing, and had recently reported that DNA sequences that alter replication timing can be used to prevent gene silencing, an important impediment for gene therapy (Fu, 2006).  We perform these analyses in established cell lines and in embryonic or hematopoietic stem cells.  We also study how cells respond to perturbation of DNA replication (Shimura, 2007). To learn more about this research, click here.

To learn more about how bio-regulatory network  control the cell cycle in normal and cancer cells,  we collaborate with a crossdisciplinary team to generate electronic molecular interaction maps, which show the behavior of cell cycle regulatory pathways during normal growth and under conditions that perturb the cell cycle (Aladjem, 2004, Kohn, 2006, 2007).  These efforts help develop bioinformatics tools that organize large collections of facts, including descriptions of networks of interacting regulatory molecules, multi-protein complexes, protein modifications (e.g. phosphorylations), etc.  To learn more about this research, click here.

Besides being a member of the Laboratory of Molecular Pharmacology, Dr. Aladjem serves as a mentor for the Howard Hughes internship program for Montgomery County Public Schools.  Dr. Aladjem helps organizing the cell cycle interest group and is also a member of the DNA repair interest group.

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Replication Studies:
 

<>Biochemical studies in mammalian cells revealed that DNA replication initiates from fixed genomic regions called replication origins or initiation regions (IRs) according to a strict temporal program.  Those initiation regions contain DNA sequences that carry genetic information essential for the initiation of DNA replication, known as replicators.  However, not all potential initiation sites in the eukaryotic genome are utilized each cell cycle and the efficiency of initiation varies between sites, suggesting that a small subset of all available replicators are activated in any single cycle.  Moreover, at some metazoan loci replication is initiated from distinct sites – so called confined sites - whereas other metazoan loci contain numerous initiation sites within an extended zone, such that each initiation site within the zone is utilized infrequently. Recent observations suggest that specification of replication sites and the timing of replication are dynamic processes that are regulated by tissue-specific and developmental cues and are responsive to epigenetic modifications.  In our laboratory we are trying to understand how replication sites and timing are specified, how replication coordinates with tissue-specific effects such as and chromatin condensation, and how the replication machinery responds to perturbation in cell cycle progression (for example, after exposure to radiation, chemical carcinogens and anti-cancer drugs). 

In previous studies (Aladjem, 1998)  we have shown that replication indeed requires specific DNA sequences and that the region between the two adult beta-like globin locu acts as a replicator.  More recently, we asked which sequences within IR dictate replicator activity. We have found that there are two independent replicators within the globin IR. Each one of these replicators can act as a DNA replication starting site. Within each replicator, we identified two sequences that cooperate to facilitate replication (Wang, 2004).    We have also dissected the requirements for initiation in each of the two replicators and identified point mutations that do not initiate replication.  We have shown that essential modules within the two replicators can combine in several ways to form functional elements that dictate the location of initiation events.  We have identified two classes of elements that need to interact to start replication and characterized the contribution of each element to initiation of DNA replication (Wang, 2006).

In the human beta-globin locus, we showed (Aladjem, 1995) that two sequence elements were essential for initiating DNA replication: the initiation region (IR), and the locus control region (LCR) residing 50 kb upstream of the IR. The LCR is also involved in regulating globin gene expression, and in the establishment of tissue and developmental specific chromatin structures. The involvement of LCR in replication implies that initiation of DNA replication may require interactions between distant sequences.  Our recent analyses suggest that LCR's function is context dependent - in the native locus it acts as an enhancer, but in other locations it can act as a silencer (Feng, 2005).  These observations suggested that the chromosomal environment may play an important role in determining origin activity.

We have also demonstrated (Aladjem, 2002) that in mouse cells, DNA replication initiates from multiple sites within the beta-globin locus.  Nevertheless, the replication timing pattern remaines similar to that observed in human cells - cells that do not express globin replicate the locus late during S-phase, whereas cells that express the protein replicate the locus early during S-phase.  This study had also shown that murine embryonic stem cells (ES cells) initiated DNA replication at specific sites, unlike some other embryonic systems in which replication initiation appeared random.  This was especially interesting because we have previously observed that ES cells do not activate some of the cell cycle controls that regulate the growth of  normal somatic cells (Aladjem, 1998).   We now use the murine system in an attempt to understand the interelations betweem the timing of DNA replication, gene expression and cell cycle control.

The human beta-globin locus is a good model to study the regulation of replication timing because this locus replicates early in S-phase in cells that express globin but replicates later in cells that do not express globin.  We found that insertion of sequences from the human beta-globin locus into a late replicating site on murine chromosome 15 alters replication timing in a sequence- and orientation-dependent manner (Lin, 2003). These observations suggest that the timing of DNA replication is controlled at the DNA sequence level, and that the sequences that modulate replication timing are distinct from the sequence elements required for replicator activity.

We have shown that inclusion of replicators, which were characterized based on their ability to dictate the location of initiation events, can also change the timing of replication and the condensation of chromatin in their vicinity.   (Fu, 2006).   This discovery is important because gene silencing is a significant impediment for gene therapy and the use of replicators can potentially improve gene therapy vectors.  We now determine which DNA sequences control replication timing and ask whether these sequences also modify the condensation state of chromatin or cooperate with replicator sequences.

We have begun to investigate how cells respond to perturbation of DNA replication.  We have shown that exposure to mild drug-induced perturbation of DNA replication, which is below the threshold of the cell cycle checkpoint response, can rapidly induce DNA breaks. In cells that contain an intact nonhomologous end joining pathway, those DNA breaks are transient and cells rapidly resume replication in the presence of the inhibitor, albeit at a slow rate. However, DNA breaks persist in cells that are deficient in components of the pathway such as DNA-PK and XRCC4; such cells are unable to resume DNA replication and activate a cell cycle checkpoint response after a mild inhibition of DNA synthesis (Shimura, 2007).

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Molecular Interaction Maps (MIMs)
 

One of the main stumbling blocks to organizing molecular knowledge is the lack of a common language that allows scientists to integrate data in a clear, standardized, and preferably computer-readable format. To that end, we implemented the Molecular Interaction Map (MIM) language, a diagrammatic annotation first proposed by Kurt Kohn, which encodes molecular information in the form of diagrams (molecular interaction maps or MIMs).  These MIMs are used to represent and analyze molecular interactions in the same way as circuit diagrams are used to trouble-shoot electronic devices.

Investigators usually describe biochemical pathways in cartoon-like diagrams, but these representations of molecular interactions are often incomplete and ambiguous.  For example, an arrow between two components could signify an increase in quantity, an increase in activity, or a modification of one molecule by the other.  In addition, enzymes in bioregulatory networks are often substrates of other enzymes, and molecules are often subject to modifications that change their binding or enzymatic capabilities.  Moreover, regulatory proteins can form multi-molecular complexes, which have different activities, depending on their composition and modifications.  Finally, each domain within regulatory molecules may have its own binding, modification, and/or enzymatic functions.  Thus, a molecule’s activity and interaction capabilities may depend on its modification state, and on the other molecules to which it may be bound.  All of these interactions must be taken into account for a full understanding of the system.

In the MIM language, we use a few defined unambiguous graphical symbols to portray each type of molecular interaction.  Each molecule is represented in a single place in a diagram, and interactions between  molecules are specified by arrows or bars at the end of connecting lines.  Because modified molecules and multi-molecular complexes may have different properties than the original molecules, the outcome of each interaction (such as a phosphorylated molecule, or a multi-molecular complex) is depicted as a circle, or “node” on an interaction line.  These nodes are treated in a way that allows them to form more interactions and extend the network. The symbols and conventions used in the language, as well as examples of MIMs, can be accessed at our website: http://discover.nci.nih.gov/mim and in articles describing the principles of the MIM language (Kohn, 2006) and their use to describe the events that lead to initiation of DNA replication(Aladjem, 2004) .

The graphical MIM language allows a simultaneous view of many interactions involving any given molecule.  It can portray competing interactions, which are common in bioregulatory networks.  An interested researcher can trace all the interactions of a given molecule from a single location.  Readers can look up a molecule in a glossary, or in the electronic (eMIM) diagrams, a mouse-click on the molecule name opens links to more information.  Each interaction is labeled with a link to an annotated description, which includes links to cited references.  The interested researcher can read the annotations to gain in-depth information on each molecular interaction, or browse the various maps to become acquainted with the general concept of how cells regulate a particular metabolic process.   For example, the eMIM depicting the early stages in DNA replication features all the possible molecular interactions between molecules involved in the process x. Additional maps represent subsets of interactions that occur during specific stages of the cell cycle and in response to cellular stress.

A major task lies ahead to compile and update maps of the major biological control systems, and to integrate them in a concise manner. We may then discern common patterns of molecular interaction logic that give bioregulatory networks their remarkable flexibility and robustness.   To elucidate the logic of signaling pathways from the multitude of molecular interactions depicted in the MIMs, we are interacting with a multidisciplinary group of researchers to develop MIM-based computer simulations.  Such tools will illustrate the processes by which cells govern DNA replication and cell cycle progression and may help understanding the perturbations in cell cycle progression that occur in cancer cells and underlie the sensitivity of these cells to anti-tumor drugs .
 
 
 
 
 

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BIBLIOGRAPHY


For a recent article published in the NIH catalyst describing Dr. Aladjem's studies, click here

                                                                                Publications in reverse chronological order


Tsutomu Shimura, Michael J. Torres, Melvenia M. Martin, V. Ashutosh Rao, Yves Pommier, Mari Katsura, Kiyoshi Miyagawa and Mirit I. Aladjem. Bloom's Syndrome Helicase and Mus81 are Required to Induce Transient Double-strand DNA Breaks in Response to DNA Replication Stress.  Journal of Molecular Biology, 375:1152-64. 2008. (Abstract-pubmed)

Aladjem, M.I. Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nat Review Genetics 8:588-600. 2007. (abstract-pubmed)

Shimura, T., Martin, M.M, Torres, M. J, Gu, C. Pluth, J. M., DeBernardi, M., McDonald, J. S. and Aladjem, M.I.  DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication. Journal of Molecular Biology 367:665-680. 2007 (Abstract - pubmed)

Seiler, J.A., Conti, C., Syed, A., Aladjem, M. I., and Pommier, Y. The Intra-S-phase Checkpoint Affects Both DNA Replication Initiation and Elongation: Single-Cell and DNA Fiber Analyses. Molecular and Cellular Biology 27:5806-18. 2007. (Abstract - pubmed)

Hong, A., Narbonne-Reveau, K., Riesgo-Escovar, Haiqing Fu, H., Aladjem, M. I., and Lilly, M.A. The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles.  EMBO Journal 26:2071-82. 2007. (Abstract - pubmed)

Kohn, K.W., Aladjem, M.I., Kim, S. Weinstein, J.N., and Pommier, Y. Depicting combinatorial complexity with the molecular interaction map notation. Molecular Systems Biology. 2:51. 2006. (Abstract - pubmed)

Wang L, Lin CM, Lopreiato JO, Aladjem MI.  Cooperative sequence modules determine replication initiation sites at the human ß-globin locus.  Hum. Mol. Genet. 15: 2613-22, 2006. Abstract - pubmed

Kohn KW, Aladjem MI.  Circuit diagrams for biological networks. Mol. Syst. Biol. 2: 2006.0002, 2006 Pubmed

Fu, H., Wang, L., Lin, C.M., Singhania, S., Bouhassira, E.E, Aladjem, M.I. Preventing gene silencing with human replicators. Nature Biotechnology, 24(5):572-6. 2006. Abstract - pubmed

Feng, Y., Desprat, D.  , Fu, H., Olivier, E., Lin, C.M., Lobell, A., Gowda, S.N., Aladjem, M. I., Bouhassira, E.E. DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells. PLoS Genetics, 2 (4) e65.  2006.   Article

Kohn, K.W., Aladjem, M.I., Weinstein, J.N., Pommier, Y. Molecular interaction maps of bioregulatory networks: a general rubric for systems biology. Mol Biol Cell. 17:1-13. 2006. Abstract - pubmed

Furuta T, Hayward RL, Meng LH, Takemura H, Aune GJ, Bonner WM, Aladjem MI, Kohn KW, Pommier Y. p21(CDKN1A) allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine. Oncogene, In Press (e-published ahead of print, 2006) Abstract - pubmed

Feng, Y.Q. Desprat, R. Fu, H. Olivier , E. Lin, C.M., Lobell, A. Gowda, S.N., Aladjem, M.I. and Bouhassira, E.E. The human ß-globin Locus Control Region can function as a silencer. Mol Cell Biol. 25:3864-74, 2005. Abstract - pubmed

Wang, J, Liu, H, Lin, C.M, Aladjem, M.I, and Epner, E.M. Targeted deletion of the chicken beta-globin regulatory elements reveals a cooperative gene silencing activity.  J Biol Chem. 280:23340-8. 2005. Abstract - pubmed

Buzina, A., Aladjem, M.I., Kolman, J.L., Wahl, G.M. and Ellis, J. Initiation of DNA replication at the human beta-globin 3' enhancer. Nucleic Acids Res. 33:4412-24. 2005 Abstract - pubmed

Aladjem, MI, and Fanning, E. The replicon revisited: an old model learns new tricks in metazoan chromosomes.  EMBO Reports 5:686-91. 2004. Abstract - pubmed

Wang, L., Lin, C.M., Brooks, S., Cimbora, D., Groudine, M. and Aladjem, M.I.   The human beta-globin replication initiation region consists of two modular independent replicators. Mol Cell Biol,24, 3373-86.  2004.Abstract - pubmed

Aladjem. MI.  The mammalian beta globin origin of DNA replication. Front Biosci. 10:1-8. 2004. abstract - pubmed

Aladjem. MI, Pasa S, Parodi S, Weinstein, JN,, Pommier, Y and Kohn, KW.  Perspective:  Molecular Interaction Maps ? a diagrammatic language for bioregulatory and signal transduction networks. ScienceSTKE 2004, pe8.  2004.Abstract-pubmed

Indig, FE., Partridge, JJ, von Kobbe, C. Aladjem, MI, Latterich, M  and Bohr, VA. Werner Syndrome Protein Directly Binds to the AAA ATPase p97/VCP in ATP-Dependent Fashion.  Journal of Structural Biology, 146:251-9.  2004 Abstract - pubmed

Lin, CM, Fu, H, Martinovsky, H, Bouhassira, E and Aladjem, M.I.  Dynamic Alterations of Replication Timing in Mammalian Cells. Current Biology13, 1019-28.  2003.Abstract - pubmed

Kohn, K.W., Aladjem MI, Pasa S, Parodi S, and  Pommier, Y.  Cell cycle control: molecular interaction map. Encyclopedia of the human genome,  Vol. 1, pp. 457-474.  2003.

Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y.   Phosphorylation Of Histone H2AX And Activation Of Mre11, Rad50, And Nbs1 In Response To Replication-Dependent DNA-Double-Strand Breaks Induced By Mammalian DNA Topoisomerase I Cleavage Complexes. Journal of Biological Chemistry, 278:20303-12. 2003. Abstract - pubmed

Nenguke, T,  Aladjem MI,  Gusella, JF, Wexler NJ, and Arnheim, N. Candidate DNA Replication Initiation Regions At Human Trinucleotide Repeat Disease Loci. Human Molecular Genetics, 12:1021-8. 2003.Abstract - pubmed

Aladjem MI, Rodewald LW, Lin CM, Bowman S, Cimbora DM, Brody LL, Epner EM, Groudine M, Wahl GM.  Replication Initiation Patterns in the beta-Globin Loci of Totipotent and Differentiated Murine Cells: Evidence for Multiple Initiation Regions. Mol Cell Biol22:442-452.2002Abstract - pubmed

Aladjem, M.I,  Spike, B. T., Rodewald, L. W., Hope, T. J., Klemm, M.,  Jaenisch, R. and Wahl, G. M.  ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage.Current Biology 8, 145-155. 1998.Abstract - pubmed

Aladjem MI, Rodewald LW, Kolman JL, Wahl GM.  Genetic dissection of a mammalian replicator in the human beta-globin locus.Science 281:1005-9. 1998.  Abstract - pubmed

Aladjem, M.I and Wahl, G. M.  Mapping replication origins by leading strand analysis in the absence of protein synthesis. Methods: companion volume to methods in enzymology13, 281-292. 1997. Abstract - pubmed

Aladjem, M.I.,  Brody, L. L.,  O'Gorman, S. and Wahl, G. M. Positive selection of FLP mediated unequal sister chromatid exchange products in mammalian cells. Molecular and Cellular Biology 17, 857-861. 1997Abstract - pubmed

Aladjem, M.I., Groudine, M., Brody, L.L., Dieken, E.M., Fournier, R. E. K., Wahl, G.M., and Epner, E.M. Participation of the human  Locus Control Region in initiation of DNA replication. Science270, 815-819. 1995. Abstract - pubmed

Kachalsky, S.G., Aladjem, M.I., Barchan, D., and Fuchs, S.,  Immunological characterization of the acetylcholine receptor binding sites from neurotoxin-resistant animals. FEBS Lett 318: 264-8.  1993.Abstract - pubmed

Fuchs, S., Barchan, D., Kachalsky, S.G., Aladjem, M.I., Ovadia, M., Kochva, E., and Neumann, D. Molecular evolution of the nicotinic acetylcholine receptor. Ann N Y Acad Sci (United States),  681: 126-39. 1993.

Oron, Y.  Vogel, R. Matus-Leibovitch, N. and Aladjem, M.I.   The hemispheric distribution of Torpedo nicotinic receptors expressed in Xenopus  oocytes. J. Basic and Clin. Physiol. and Pharmacol. 4, 181-197. 1993.Abstract - pubmed

Aladjem, M.I., and Lavi, S.,  The mechanisms of carcinogen-induced gene amplification in vivo and in vitro. Mutation Research 276: 339-344.  1992.Abstract - pubmed

Aladjem, M.I., and Lavi, S.,  Carcinogen induced activation of SV40 gene expression in a semi-permissive environment. Virology 189: 493-499.  1992.Abstract - pubmed

Aladjem, M.I., and Lavi, S. Carcinogen-induced gene amplification and gene expression. In: Rossman, R.G. (ed): Induced effects of genotoxic agents in eukaryotic cells. pp. 113-123. Taylor and Francis, Washington, D.C. 1992.

Lavi, S., Hassin, D., Aladjem, M.I., Ophir, Z., and Karby, S.  "U turn replication at arrested forks leads to the generation of extrachromosomal inverted repeats.  In: R. E. Kellems (ed):Gene amplification in mammalian cells: techniques and applications.  Marcel Decker, N.Y.1992.

Aladjem, M.I., Koltin, Y., and Lavi, S.  Enhancement of copper resistance and CupI amplification in carcinogen treated yeast cells. Mol. Gen. Genet.211: 88-94.  1988.Abstract - pubmed

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SYMPOSIUM: CELL CYCLE REGULATION

Balcony A, Natcher Auditorium, May 9th, 2006

Sponsored by the Cell Cycle Interest Group

Organized by Munira A. Basrai and Mirit I. Aladjem


(For a poster describing the event, click here)


10:00 am  Keynote address: Yosef Shiloh
Sackler School of Medicine, Tel Aviv University, Israel
ATM: Fine-Tuning of the DNA Damage Response

11:00 am Mary Ellen Perry (NCI) 
 Physiological functions of MDM2.

11:20 am Jennifer Seiler (NCI) 
DNA replication is inhibited selectively in late-S phase cells by the topoisomerase 1 inhibitor camptothecin

11:40 Akira Motegi (NHGRI)
 Human SHPRH suppresses genomic instability through PCNA polyubiquitination
 
(Lunch break 12:00 – 1:30 pm)

1:30  pm John Weinstein (NCI) 
High throughput integromic profiling of cancer cells:  genomic, proteomics and bioinformatics”

1:50 pm Sohyoung Kim (NCI)
 Molecular modeling of cell cycle interactions

2:10  pm Chiara Conti (Pasteur Institute, Paris, France)
Single molecule analyses of DNA replication

2:40 pm Joseph Campbell (NIDDK)
 What weird nuclei can tell us about domains of the nuclear membrane

3:00 pm Mary Lilly (NICHD) 
The Cyclin-Dependent Kinase Inhibitor Dacapo promotes the licensing of DNA replication origins during Drosophila endocycles

3:20 pm Alex Sturnikov (NICHD)
Spindle checkpoint monitors condensin function".

3:40 pm Cyril Berthet (NCI)
Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation