Each dividing cell must undergo precise DNA replication to ensure faithful transmission of its DNA to its daughter cells.  In eukaryotic cells, replication occurs according to a tightly regulated temporal program with some regions being replicated earlier and others later.  This timing program significantly correlates with many genomic features such as mutation rates and is thus an important mechanism for understanding changes occurring to the DNA both in inheritance and in disease such as cancer.  We have established novel statistical and computational methods to analyze replication timing profiles of different cells to better understand how replication timing works and how the changes in this program affect the genome in both healthy and disease states.

The following image shows an example of replication timing plots of different MEF samples.

 

 

Though usually both alleles of a genes replicate at the same time, occasionally the same gene will replicate each allele at different times resulting in asynchronous replication. Asynchronous replication is assumed to be an important regulatory feature to allow the cell to express only one copy of the allele.  This occurs by imprinted genes as well as by other mono-allelically expressed genes.  Despite the importance of this mechanism, to date, no comprehensive studies have determined the genome wide map of asynchronous replication of a clonal cell line.  Using F1 mice, from Castaneous and C57BL/6 mouse offspring and separating alleles according to specie-specific SNPs, we have created a genome wide map of asynchronous replication and are characterizing asynchronous regions in order to further understand the mechanisms and regulation of asynchronicity as well as its role in disease.

 

Deregulated replication timing has been discovered in a number of cancers and influences the pattern of genomic instability.  To better understand the function and importance of these changes, we are studying replication timing alterations occurring during the development of Ras induced cancer.  We have created inducible cancerous cell systems in which we are measuring the replication timing along cancer progression in order to elucidate the connection between the replication timing program and cancer.  With these results, we hope to shed light on important mechanisms which lead to the accumulation of mutations in cancer.

 

Structural variations of cellular DNA are known to distinguish between different species or cell types.  We are currently investigating the possibility that unique chromosomal variations also exist between different tissue types.  By analyzing existing WGS data of different healthy tissues, we are studying the frequency of different structural variations in a tissue specific manner. 

DNA replication begins at the many origins or replication spread throughout the DNA.   It is known that in each cell cycle only some of the potential origins of replication present in each cell are activated.  However, to date, it is impossible to obtain accurate information about the efficiencies of individual origin of replication.

In order to address this challenge, we collaborated with Professor Yuval Ebenstein (Tel-Aviv University) who developed optical mapping based methods for mapping individual large DNA molecules to the genome. Coupling optical mapping with labeling newly replicated DNA (using the IdU analogue) allows the localization of the regions that undergo replication.

We are currently using this method for mapping yeast early origins while obtaining information about the efficiency of each origin.

 

DNA molecules (green) labeled with IdU (red).

 

DNA molecules (Blue) labeled with optical barcodes (red)

DNA replication occurs in a certain order: certain regions of the genome are replicated early in S phase whereas other regions replicate late in S phase.

We are comparing DNA replication in regions that replicate early in S phase versus regions that replicate late in S phase. We are using a combination of FACS and DNA combing to measure features such as the replication fork rate and the inter origin distance. We also compare these features in cells exposed to mild replication stress (hydroxyurea or aphidicolin), to compare the stress response mechanisms that are used throughout S phase. For example, it is thought that the accuracy of DNA replication is higher in early S compared to late S.

We are also interested in the features of DNA replication in normal and stressed conditions in cell that express mutant proteins in proteins that are known to regulate different aspects of replication. For example, Rif1 was recently identified as a regulator of the time of replication. Depletion of the Rif1 protein has been demonstrated to disrupt the normal pattern of replication timing in cells from several organisms. We are using a series of cell lines expressing Rif1 proteins that contain a point mutation to examine which protein-protein interactions are required for the function of Rif1 in regulating the time of replication.

We are using a similar approach to study the role of the cohesin complex in regulating DNA replication during S phase and in response to stress conditions

One of the fascinating question in the cell cycle field is how the epigenetic information is preserved from mother to daughter cells. During mitosis the chromosomes are condensed, transcription ceases and many proteins fall off the chromosomes. Nevertheless, the information regarding the identity of each genomic region is preserved and transcriptional activity is rapidly restored after mitosis. This necessitates precise epigenomic markings, which are likely preserved during mitosis despite the radical changes to the chromatin. How is this epigenomic information stored during mitosis?

To better understand the role of mitotic histone modifications in mitotic bookmarking, we investigated the global dynamics and genomic organization of histone modifications in mitosis and in interphase using advanced proteomics and genomic tools. Altogether, integration of these data provided two major insights: First, the epigenomic landscape does not change considerably during mitosis. Second, nucleosomes surrounding the Nucleosome depleted regions of promoters, enhancers, and insulators are repositioned and modified during mitosis.

The following image shows an example of the distribution of several histone modifications in interphase (blue) and mitosis (red). Note that in spite of the global condensation there are hardly any changes in histone modifications, suggesting that the epigenetic memory can be carried through mitosis by histone modifications.