Mirit I. Aladjem, Ph.D.
Developmental Therapeutics Branch
Building 37, Room 5068D
Bethesda, MD 20892-4255
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.
The challenge of understanding DNA replication: 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.
Research strategy: Signals from cell cycle regulatory networks ultimately converge on chromatin to regulate the early steps of DNA replication. We aim to understand how the location and the timing of replication events are linked to particular modifications on chromatin and how 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 identify proteins that bind such sequences to determine where and when replication initiates. Second, we use whole-genome sequencing and replication imaging approaches to study the dynamics of cellular DNA synthesis and determine how replication patterns respond to alterations in gene expression, chromatin modifications and drugs that perturb replication.
Role of replication origins: Within eukaryotic cells, genome duplication initiates at multiple sites on each chromosome (replication origins). Replication initiation events in diploid mitotic cells proceed in a precise order and are strictly regulated by a series of cell cycle checkpoint signaling pathways. These regulatory constraints, however, are often relaxed in cancer cells. Understanding the molecular events that precede DNA replication at the chromatin level is crucial if we are to fully understand cell growth. Critical information about this process is missing because protein complexes that initiate chromosomal replication seem to bind DNA indiscriminately. To gain a complete understanding of the DNA replication process we must resolve how this non-specific DNA binding translates into highly coordinated replication.
Our studies are based on the hypothesis that sequence-specific signaling molecules associate with replication initiation sites on chromatin where they modulate the local activity of the ubiquitous replication machinery and dictate both the location and timing of replication initiation events. To test this hypothesis, we characterize protein-DNA interactions at replication initiation sites and identify interactions that play regulatory roles in the DNA replication process.
Protein-DNA interactions at replication origins: We use two approaches to characterize DNA-protein interactions at replication initiation sites. The first approach utilizes distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication. We have initially identified and characterized replicator sequences in mammalian cells, using the human beta-globin locus as a model (Aladjem, Science 281, 1005. 1998). We now use replicators as bait to isolate protein complexes that potentially regulate replication. In recent studies we have identified two discrete DNA-protein complexes within one replicator element. One of these complexes includes chromatin remodeling proteins that determine both replication timing and transcriptional activity (Huang, Mol Cell Biol. 31:3472-84; 2011). Another complex includes RepID, a member of the DDB1-Cul4-associated-factor (DCAF) family, which binds a subset of replication initiation sites and is required for replication at those sites (Zhang, Nat Commun. 8;7:11748; 2016). Our studies have demonstrated that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. We have characterized RepID interactions with other proteins, identified RepID protein partners using a non-biased approach and pinpointed protein domains within RepID that facilitate DNA-protein and protein-protein interactions. Our analyses demonstrate that RepID binding origins require RepID for initiation of DNA replication, providing the first example of a site-specific interaction that determines the initiation of DNA replication on a group of metazoan replication origins.
Whole-genome chromatin patterns at replication origins: The second approach involves developing tools to map replication initiation sites throughout the genome, and using these tools to analyze DNA replication in the context of chromatin modifications and transcriptional activity. The developed methods involve massively parallel sequencing and single-fiber imaging of replication fork progression. These procedures allow us to study the dynamics of DNA replication at the whole-genome level. Using this methodology we can test whether groups of replication initiation sites share specific properties - for example, if they associate with a particular chromatin feature. We can also identify groups of initiation sites that respond in a similar fashion to a cellular challenge, and test whether distinct groups of replication initiation sites are regulated through association with particular proteins (such as RepID). We have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines (Martin, Genome Res. 21:1822-32, 2010; Smith, Epigenetics and Chromatin 9:18, 2016). To facilitate these studies, we have developed a web-based tool (Coloweb; Kim, BMC Genomics 16:142. 2015) to help decipher the relationships among RepID binding sites and epigenetic features. This tool is available to the community to support bioinformatics characterization of DNA-protein interaction loci. We use genome-wide data to identify DNA and histone modifications that associate with replication initiation events. For example, we observed strong associations between replication initiation and both DNAse hypersensitive sites and dimethylated histone H3 lysine 79, which exhibits a dynamic cell cycle distribution (Fu, PLoS Genet. 9:e1003542, 2013).
Modulation of DNA replication: Our recent comprehensive analysis of chromatin modifications associated with DNA replication origins (Smith, Epigenetics and Chromatin 9:18, 2016) demonstrated that replication origin usage varies with tissue type, with distinct modifications associated with cell-type specific replication origins. In a collaborative study, we have used phased allele-specific analyses of replication origins to decipher the sequence requirements for replication initiation (Bartholdy, Nature Communications 6:7051. 2015). Anti-cancer drugs often target DNA replication and/or interfere with cell cycle signaling, and the precise nature of the cell cycle defect in a particular cancer often influences the cancer cell's susceptibility to specific anti-cancer therapy. Combining nascent strand abundance sequencing and single fiber analyses, we asked how particular replication and repair pathways affect the pace and frequency of DNA replication. We observed that a DNA repair endonuclease, Mus81, modulates the pace of DNA replication in the absence of exogenous stress and that its presence is essential to help cells restore DNA synthesis in the presence of drugs that slow replication (Fu, Nature Communications 6:6746. 2015). In the future we will investigate how protein-DNA interactions that are required for DNA replication are modulated in response to environmental challenges and anti-cancer drugs.
Aladjem, M.I., Rodewald, L.W., Kolman J. L. and Wahl, G.M. Genetic dissection of a mammalian replicator in the human beta-Globin locus. Science 281, 1005. 1998.
Bartholdy B, Mukhopadhyay R, Lajugie J, Aladjem MI, Bouhassira EE. Allele-specific analysis of DNA replication origins in mammalian cells. Nat Commun.6:7051. 2015.
Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nature Communications 6:6746. 2015.
Fu H, Maunakea AK, Martin MM, Huang L, Zhang Y, Ryan M, Kim R, Lin CM, Zhao K, Aladjem MI. 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. 2013.
Huang L, Fu H, Lin CM, Conner A, Zhang Y, Aladjem MI. Prevention of transcriptional silencing by a replicator-binding complex consisting of SWI/SNF, MeCP1 and hnRNP C1/C2. Molecular and Cellular Biology 31:3472-84. 2011.
Kim R, Smith OK, Wong WC, Ryan AM, Ryan MC, Aladjem MI. ColoWeb: A Resource for Analysis of Colocalization of Genomic Features. BMC Genomics 16:142.2015.
Martin MM, Ryan M, Kim R, Zakas AL, Fu H, Lin CM, Reinhold WC, Davis SR, Bilke S, Liu H, Doroshow JH, Reimers MA, Valenzuela MS, Pommier Y, Meltzer PS, Aladjem MI. Genome-wide depletion of replication initiation events in highly transcribed regions. Genome Research 21: 1822-1832. 2011.
Smith OK, Kim RG, Fu H, Martin M, Utani K, Zhang Y, Marks AB, Lalande M, Chamberlaine S, Libbrecht MW, Bouhassira EE, Ryan MC, Noble WC, Aladjem MI. Distinct Epigenetic Features of Differentiation-Regulated Replication Origins. Epigenetics and Chromatin 9:18. 2016.
Zhang Y, Huang L, Fu H, Smith OK, Lin CM, Utani K, Rao M, Reinhold WC, Redon CE, Ryan M, Kim RG, You Y, Hanna H, Boisclair Y, Long Q, Aladjem MI. A Replicator-Specific Binding Protein Essential For Site-Specific Initiation of DNA Replication in Mammalian Cells. Nat. Commun. 7:11748. 2016.
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.
Utani K, Fu H, Jang SM, Marks AB, Smith OK, Zhang Y, Redon CE, Shimizu N, Aladjem MI. Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability. Nucleic Acids Res. 2017;45(13):7807-7824.
Smith OK, Kim R, Fu H, Martin MM, Lin CM, Utani K, Zhang Y, Marks AB, Lalande M, Chamberlain S, Libbrecht MW, Bouhassira EE, Ryan MC, Noble WS, Aladjem MI. Distinct epigenetic features of differentiation-regulated replication origins. Epigenetics Chromatin. 2016;9:18.
Zhang Y, Huang L, Fu H, Smith OK, Lin CM, Utani K, Rao M, Reinhold WC, Redon CE, Ryan M, Kim R, You Y, Hanna H, Boisclair Y, Long Q, Aladjem MI. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells. Nat Commun. 2016;7:11748.
Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nat Commun. 2015;6:6746.
Fu H, Maunakea AK, Martin MM, Huang L, Zhang Y, Ryan M, Kim R, Lin CM, Zhao K, Aladjem MI. 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. 2013;9(6):e1003542.
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This page was last updated on September 12th, 2018