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Mirit I. Aladjem, Ph.D.

Portait Photo of Mirit Aladjem
Developmental Therapeutics Branch
Head, DNA Replication Group
Senior Investigator
Center for Cancer Research
National Cancer Institute
Building 37, Room 5068D
Bethesda, MD 20892-4255
Phone:  
301-435-2848
Fax:  
301-402-0752
E-Mail:  
aladjemm@mail.nih.gov

Biography

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 and a Leukemia Society Special Fellow at the Salk Institute in La Jolla, California. Dr. Aladjem joined the Laboratory of Molecular Pharmacology/Developmental Therapeutics Branch in October 1999 and was appointed a Senior Investigator in 2007. Dr. Aladjem's studies focus on cellular signaling pathways that modulate chromatin to regulate chromosome duplication and cell cycle progression.

Research

Goal
The broad goal of the DNA Replication Group at the Developmental Therapeutics Branch is to understand cellular networks that signal to and from chromatin to modulate DNA replication. Since many regulatory feedback pathways are deregulated in cancer cells, the results of these studies will help our understanding of cancer biology and elucidate how normal and cancer cells regulate DNA replication. 


Background

Loss of genetic control of DNA replication is a hallmark of cancer cells. Protein signaling pathways that reulate cell growth converge on molecular events that facilitate DNA replication. Replication regulatory pathways can provide good targets for synthetic lethality approaches that specifically kill cancer cells, but replication problems that go undetected can affect genomic integrity, triggering genomic instability that eventually might result in cancer drug resistance. Hence, many anti-cancer drugs target various aspects of DNA replication and the effectiveness of such drugs critically depends on the nature of the lesions affected in particular cancers. To understand how cells regulate their growth, we employ biochemistry, molecular genetics and bioinformatics to ask how cells determine where and when DNA replication starts. As part of the Developmental Therapeutic Branch, we are also involved in collaborative studies aimed to develop better ways to describe regulatory feedback networks that modulate cell cycle progression and the response of cells to anti-cancer drugs. 


DNA Replication Analyses
Because signals from cell cycle regulatory networks ultimately converge on chromatin, we aim to understand how the location and the timing of replication events are linked to particular modifications on chromatin and how chromosome replication coordinates with other chromatin transactions such as transcription, DNA repair and chromosome condensation. To that end, we take two complementary approaches. First, we use biochemical and genetic approaches to dissect DNA sequences that facilitate replication and proteins that bind such sequences. This effort is expected to advance our understanding of processes that govern how cells determine where and when replication would start. Second, we use massively parallel sequencing and DNA fiber imaging approaches to study the dynamics of DNA replication throughout the genome. Those studies aim to determine how replication patterns respond to alterations in gene expression, chromatin modifications and drugs that perturb replication.
Genetic analyses of DNA Replication.
We study DNA sequences (termed replicators) that facilitate initiation of DNA replication at their endogenous chromosomal sites or when they are removed from their endogenous location and transferred to ectopic chromosomal sites. In previous studies, we have identified replicator sequences in mammalian cells and dissected the genetic determinants essential for replicator activity. These studies were performed in depth in a single genomic locus, the human beta globin locus on chromosome 11 (Aladjem, Rodewald et al. 1998; Wang, Lin et al. 2004; Wang, Lin et al. 2006). Our observations suggest that that epigenetic processes play a role in determining if and when a particular replicator will start replication during each S-phase (Fu, Wang et al. 2006, reviewed in Aladjem, 2007; Conner and Aladjem, 2012). We have identified protein-DNA interactions in replicator sequences. In particular, we have demonstrated distinct functions two discrete DNA-protein complexes we have identified at the human beta globin locus. One complex includes chromatin-remodeling proteins, affects replication timing and transcriptional activity and mediates an interaction between the replicator and a distal locus control region (Huang, Fu et al. 2011). The other complex interacts with the pre-replication complex and is essential for initiation of DNA replication. These findings imply that the locations of initiation events depend on interactions of cell cycle regulatory proteins with sequence modules that reside within potential replicators and that the proteins that regulate initiation function in a cooperative and combinatorial manner. Such interactions may underlie the variable use of initiation sites observed in mammalian chromosomes and determine the timing of replication. 

Whole-genome Replication analyses.
We have determined the genome-wide distribution of replication initiation events in several human cancer cells (Martin, Ryan et al. 2011) and in non-cancerous erythroblasts (Mukhopadhyay et al, 2014). The dataset created by the genome-wide studies encompasses the locations of replication initiation sites throughout the entire non-repetitive genomes of the analyzed cells. We found that the frequency of replication initiation events increased in genomic regions that were transcribed in moderate levels but that initiation frequency was reduced in genes with high transcription rates. In concordance, high-resolution mapping showed that replication initiation events were excluded from promoter regions and enriched immediately downstream of transcribed promoters. We also found that the frequency of initiation events was affected by chromatin condensation and methylation at CpG sequences. In recent studies, we have identified chromatin modification patterns characteristic of distinct groups of replication origins (such as replication origins that are active only at specific tissues or are activated at distinct cell cycle phases). One particular modification, methylation of histone H3 on lysine 79, plays a role in restricting replication to a single round of duplication each cell cycle (Fu et al, 2013). These findings allow us to propose a model suggesting a role for protein-DNA interactions and chromatin modifications at replicator sequences in coordinating replication, transcription and chromatin condensation. 

Analyses of Perturbed Replication
Our previous studies have identified a new pathway involved in the cellular response to replicative stress. We showed that low non-toxic doses of replication inhibitors decelerate replication by a mechanism involving the cancer-predisposing protein BLM helicase, Mus81 nuclease and ATR kinase. In early stages of the pathway, inhibitors induce transient DNA breaks that are rapidly repaired by the non-homologous end-joining (NHEJ) pathway involving DNA-PK and XRCC4. Rapid repair of the DNA breaks prevents cell cycle arrest despite minor changes in the rate of replication fork progression (Shimura, Martin et al. 2007; Shimura, Torres et al. 2008). We have also collaborated with Dr. Pommier's group in DTB to characterize the response of cancer cells to drugs that perturb DNA replication (Seiler, Conti et al. 2007; Conti, Leo et al. 2010) and to determine the role of a nuclease, Mus81, in generating DNA breaks following perturbation of DNA replication by campthotecin, a DNA topoisomerase inhibitor (Regairaz et al. 2011). The combination of genome-scale sequencing of replication initiation sites and single fiber analyses can provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication..
Integrative studies and Molecular Interaction Maps
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 that circuit diagrams are used to trouble-shoot electronic devices. 

For this project, we closely collaborate with Dr. Kohn. We have developed and released several tools for creating and editing MIM diagrams (Luna, Karac et al. 2011; Luna, Sunshine et al. 2011; Chandan et al. 2011). These tools should make it easier for developers to build MIM-related software, users to create and edit MIM diagrams. We have also participated in international collaborations to develop the systems biology is developed by an international consortium with our participation (Le Novere, Hucka et al. 2009; van Iersel et al. 2012). 
In a separate line of study, we use MIMs as a basis for mathematical modeling of cellular regulatory networks in an effort to shed light on basic feedback mechanisms that modulate cell proliferation (Kim, Aladjem et al. 2010). We have created an extensive map describing bioregulatory interactions involving the SIRT1 deacetylase Luna et al., 2913) and a subset of this map was also employed in mathematical modeling and simulations.
References 

Aladjem, MI. (2007). Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nat Rev Genet 8(8): 588-600. 


Aladjem MI, Rodewald LW, et al. (1998). Genetic dissection of a mammalian replicator in the human beta-globin locus. Science 281(5379): 1005-1009. 


Chandan K, van Iersel MP, Aladjem MI, Kohn KW, and Luna A. (2012). PathVisio-Validator: a rule-based validation plugin for graphical pathway notations. Bioinformatics 28: 889-90. 


Conner AL, Aladjem MI. (2012). The chromatin backdrop of DNA replication: Lessons from genetics and genome-scale analyses. Biochim Biophys Acta 1819: 794-801. 


Conti C, Leo E, et al. (2010). Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins, and induces DNA damage. Cancer Res 70(11): 4470-4480. 


Fu H, Maunakea AK, Martin MM, Huang L, Zhang Y, Ryan M, Kim R, Lin CM, Zhao K, Aladjem MI.2013. Methylation of histone H3 on lysine 79 associates with a group of replication origins and helps limit DNA replication once per cell cycle. PLoS Genet 9: e1003542
Fu H, Wang L, et al. (2006). Preventing gene silencing with human replicators. Nat Biotechnol 24(5): 572-576. 


Huang L, Fu H, et al. (2011). Prevention of transcriptional silencing by a replicator-binding complex consisting of SWI/SNF, MeCP1, and hnRNP C1/C2. Mol Cell Biol 31(16): 3472-3484. 


Kim S, Aladjem MI, et al. (2010). Predicted functions of MdmX in fine-tuning the response of p53 to DNA damage. PLoS Comput Biol 6(2): e1000665. 


Le Novere N, Hucka M, et al. (2009). The Systems Biology Graphical Notation. Nat Biotechnol 27(8): 735-741. 

Luna A, Karac EI, et al. (2011). A formal MIM specification and tools for the common exchange of MIM diagrams: an XML-Based format, an API, and a validation method. BMC Bioinformatics 12(1): 167. 


Luna A, Sunshine ML, et al. (2011). PathVisio-MIM: PathVisio plugin for creating and editing molecular interaction maps (MIMs). Bioinformatics 27(15): 2165-2166. 


Luna A, Aladjem MI and Kohn KW. SIRT1/PARP1 Crosstalk: connecting DNA damage and metabolism. Genome Integrity 4:6. 2013.
Martin MM, Ryan M, et al. (2011). Genome-wide depletion of replication initiation events in highly transcribed regions. Genome Res. 21(11):1822-32. 


Mukhopadhyay R, Lajugie J, Fourel N, Selzer A, Schizas M, Bartholdy B, Mar J, Lin CM, Martin MM, Ryan M, Aladjem MI, Bouhassira EE.2014. Allele-Specific Genome-wide Profiling in Human Primary Erythroblasts Reveal Replication Program Organization. PLoS Genet 10: e1004319
Seiler JA, Conti C, et al. (2007). The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses. Mol Cell Biol 27(16): 5806-5818. 


Shimura T, Martin MM, et al. (2007). DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication. J Mol Biol 367(3): 665-680.
Shimura T, Torres MJ, et al. (2008). Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress. J Mol Biol 375(4): 1152-1164. 


van Iersel MP, Villeger AC, Czauderna T, Boyd SE, Bergmann FT, Luna A, Demir E, Sorokin A, Dogrusoz U, Matsuoka Y, Funahashi A, Aladjem MI, Mi H, Moodie SL, Kitano H, Le Novere N, Schreiber F. (2012) Software support for SBGN maps: SBGN-ML and LibSBGN. Bioinformatics 28:2016-2021. 

Wang L, Lin CM, et al. (2004). The human beta-globin replication initiation region consists of two modular independent replicators. Mol Cell Biol 24(8): 3373-3386. 


Wang L, Lin CM, et al. (2006). Cooperative sequence modules determine replication initiation sites at the human beta-globin locus. Hum Mol Genet 15(17): 2613-2622.



This page was last updated on 6/23/2014.