Research Lab for Chromatin and Aging
Our lab is interested in understanding the dynamics of heterochromatin during cellular and developmental processes and how these contribute to the process of aging in different tissues. We combine genomic tools and various molecular techniques as well as live cell imaging in our studies. We take advantage of several model systems- including cell lines, mice and fission yeast in our experiments, and also study samples from the clinic to show the clinical relevance of our findings.
One of the ways by which big and drastic changes in behavior of cells can occur is by regulation of chromatin. Chromatin is defined as the assembly of DNA and the proteins which bind to it such as histones, transcription factors and structural components needed for its maintenance. Many DNA binding proteins, and in particular histones, are modified by enzymes which add chemical modifications such as methylation, phosphorylation, acetylation and ubiqutination, thus changing the physical and chemical properties of these proteins and of the chromatin, accessibility to other binding proteins, and the RNA expression of the genes which are bound by the histones.
In general terms, chromatin can be subdivided into two types of chromatin: the more accessible, gene rich euchromatin, and the more compact, non-accessible, gene-poor heterochromatin. Heterochromatin and its composition are important for the regulation of gene expression and for mediating transcriptional silencing. In addition, it is also critical for the stability of the genome. Previous studies have shown that the type of heterochromatin found at genomic maintenance related loci such as telomeres, centromeres and retrotransposons is critical for their functions. Heterochromatin regulation and dynamics may become especially important during the cell cycle, when the expression of many genes, as well as the status of centromeres and telomeres is constantly changing- affecting genomic stability. The regulation of heterochromatin configuration is the focus of our interest in the lab.
The effect of heterochromatin changes with age in the reproductive system:
Aging affects all cells, but is prominent in the reproductive system. Oocytes age early, becoming aneuploid, thus limiting female reproductive age. Aged oocytes display phenotypes involving chromosome non-disjunction, causing genetic abnormalities such as Down’s syndrome. Spermatocytes age later than oocytes, but still show a reduction in fertility and an age-dependent rise in genetic diseases such as autism and schizophrenia. It has now become evident that age-related changes in heterochromatin contribute significantly to the etiology of aging. Using molecular biology tools together with microscopy, we will decipher the effect of heterochromatin changes on the aging of gametes. We will determine which molecular pathways and players in heterochromatin are at play when gametes age, and if these pathways can be modified to delay the aging process in the reproductive system. We will use a three-pronged approach: We first aim to characterize the changes in heterochromatin during aging of spermatocytes and oocytes, using a myriad of genomic and microscopy tools for this aim, including cutting-edge technologies like single cell methylation sequencing. Second, we will study the mechanism of age-related aging in the reproductive system in depth. We will determine the causal relationship between heterochromatin dynamics and centromere binding proteins in oocytes, and develop a yeast model to study the heterochromatin-related mechanistic questions that affect the nuclear structure and cell cycle arrest characteristic to meiosis. Finally, we aim to identify the rules governing the trans-generational inheritance of heterochromatin epigenetic changes. We will identify genomic regions where parental age effects the next generation, and use bioinformatics to decipher the molecular rules governing this mode of inheritance.
Together, these experiments will yield a comprehensive picture of how aging affects heterochromatin, and whether molecular tools can prevent any ill effects.
The Link between Genomic instability, Abnormal Meiotic Gene Expression and Tumor Formation:
Oral Squamous Cell Carcinoma as a model:
Cancer cells have a propensity to re-arrange their genome. Tumors tend to lose or gain whole chromosomes and re-arrange large genomic regions. When this process occurs continuously, the tumor becomes chromosome instable (CIN). CIN tumors are more aggressive and often harder to treat. In this project we aim to identify a new mechanism for CIN. It was previously shown that many tumors express high levels of genes which participate in meiosis. Meiosis is a process which occurs only in the reproductive system and is responsible for the reduction division which turns eggs and sperm into haploid cells (containing one copy of each chromosome). Since chromosome instability is built-in during meiosis (by the processes of homologous recombination and mono-orientation), the overexpression of meiosis-specific genes could in principle cause the tumor to become CIN.
We will carry out a bioinformatics survey of existing tumor data, together with an analysis of samples of oral cancer from the clinic in order to determine the correlation level between meiotic gene expression and CIN.
We will also mechanistically study the process, and overexpress single meiotic genes in cell lines, and measure the CIN in them, and also use the genetically trackable fission yeast model to see the effect of meiotic gene expression on chromosome stability.
This new mechanism could lead to the identification of markers and drug targets to diagnose and treat CIN tumors.
The effect of high fat diet on the gut epigenome and risk for cancer:
Diet has a profound impact on tissue regeneration, aging, and disease in mammals. However, the mechanisms through which diet perturbs stem and progenitor cell biology and leads to diseases, such as cancers are poorly understood. With the rise of obesity in the US population – more than 1 in 3 adults are obese – the relationship between diet, stem cell biology, and cancer takes on great importance.
Focused on the gut, we find that a pro-obesity high fat diet (HFD) augments the numbers and niche-independent function of Lgr5+ intestinal stem cells (ISCs) of the mammalian intestine and enhances the self-renewal potential of intestinal organoid bodies (ex vivo mini-intestines). In preliminary studies we find that a HFD engages a stable cellular state in intestinal stem and progenitor cells that persists even after reversion to a normal chow diet. Specifically, we will test the hypotheses that the effects of a HFD persist upon reversion to a normal chow diet, that intestinal stem and progenitor cells undergo changes in their DNA methylation state, that age-related methylation accelerates the effects of a HFD, and that genetic or pharmacologic interventions that target the DNA methylation apparatus can counter these effects of a HFD.
These experiments will enable the understanding of how DNA methylation may facilitate obesity related health problems, and how the epigenetic landscape may be modified by diet. The results of these experiments may identify novel interventions for colon cancer prevention and treatment as well as generate recommendations for keeping a healthy epigenome through diet management.
Cellular re-cycling: cell cycle re-entry after arrest – the chromatin angle:
Cells respond to cues from their environment in many ways. One of type of response is to regulate their exit from the cell cycle. In fact, most cells in a tissue at any given time are not actively cycling, and are found in a quiescent state termed G0. This type of response is not restricted only to multicellular organisms. Unicellular organism such as yeast and other fungal pathogens can also exit the cell cycle and enter a quiescent state. Many pathogens find their way into their target sites in this state. Cancer stem cells, which may initiate a tumor or metastasize, are also found in G0 most of the time.
When the environment changes, the cell may decide to enter the cell cycle again. Using this type of mechanism cells initiates a tumor, pathogens start an infection, and cells expand the cell number in a tissue. The mechanism by which the cells re-enter the cell cycle is still obscure despite its obvious importance.
One possible mechanism by which cells may regulate their cell cycle re-entry is through chromatin regulation. Global and local chromatin changes have been found to be associated with a quiescent state in fibroblasts and with virulence in the pathogenic fungus Candida albicans. Our preliminary results suggest that chromatin regulation is also part of cell cycle re-entry in the fission yeast Schizosaccharomyces pombe. Despite these initial results, information about chromatin regulation of cell cycle regulation and re-entry is lacking.
In this project, we will expand the known information about cell cycle re-entry. We will use three cellular systems: fission yeast, human fibroblasts and the pathogen C. albicans, in order to study chromatin regulation of this process. We will arrest the cell cycle in each of the system, re-initiate it, and analyze chromatin during the re-entry process. We will use ChIP coupled with next generation sequencing (ChIP-seq), to analyze the genome-wide location of chromatin binding proteins and histone modifications, for which we find evidence of their involvement in the re-entry process. In addition, we intend to use live-cell imaging in order to look for pan-nuclear changes in chromatin structure during the re-entry process. Using these tools, we intend to compile a list of suspected genes involved in this process. We will then analyze the behavior of mutants in these genes, and identify the mechanism by which chromatin regulation occurs during cell cycle re-entry. Finally, we will use chromatin drugs in order to find possible targets that can be used to inhibit the process of re-entry, and possibly target pathogens or cancer stem cells.
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Dr. Michael Klutstein
Dr. Eli Reich
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1. Klutstein M, Fennell A, Fernandez-Alvarez A and Cooper J P (2015) The telomere bouquet regulates meiotic centromere
assembly – Nature Cell Biology, doi: 10.1038/ncb3132.
2. Klutstein M and Cooper J P (2013) ‘the chromosomal courtship dance’- homolog pairing in early meiosis, Current Opinion in
Cell Biology, 26, 123-131.
3. Valente L, Dehe PM, Klutstein M, Aligianni S, Watt S, Bahler J and Cooper J P(2013) Myb-domain protein Teb1 controls
histone levels and centromere assembly in fission yeast- EMBO Journal, 32 (3), 450-460.
4. Klutstein M, Siegfried Z , Gispan A, Frakash-Amar S, Zinman G, Bar-Josheph Z Simchen G and Simon I. (2010) Combination
of genomic approaches with functional genetic experiments reveals two modes of repression of yeast middle-phase meiosis
genes- BMC Genomics,11, 478.
5. Klutstein M, Xaver M, Shemesh R, Zenvirth D, Klein F and Simchen G.(2009) Heterozygous zip1 mutants of Saccharomyces
cerevisiae reveal additional roles in meiosis –Molecular Genetics and Genomics-282(5), 453-462.
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We are located in the Ein Kerem Campus of the Hebrew University, Jerusalem.
Michael Klutstein, PhD.
Faculty of Dental Medicine
Hebrew University – Hadassah
POB 12272, Jerusalem 91120, Israel
Tel (mobile): +972-584786952
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