Dipanjan Chowdhury, Ph.D.

Research

DNA repair- a molecular perspective

DNA damage occurs constantly from endogenous (e.g. reactive oxygen species, metabolic byproducts, DNA replication and recombination) and exogenous (e.g. genotoxic chemicals, ionizing radiation and UV irradiation) sources.  To survive DNA damage, cells have to rapidly sense the DNA break and proliferating cells have to stop dividing via cell cycle check points.  The DNA lesion is repaired using appropriate machinery or irreparable DNA breaks induce programmed cell death.  Therefore, defects in DNA repair or cell cycle checkpoint components lead to genomic instability resulting in chromosomal translocations and unrestricted cell growth.

Genomic Stability and Lymphoma: Chromosomal translocations underlie many human lymphomas, sarcomas, and epithelial tumors. The most common translocation is the juxtaposition of an oncogene with an antigen receptor gene causing transcriptional dysregulation of the oncogene.. Major translocations identified in non-Hodgkins lymphoma include those fusing BCL2 with immunoglobulin heavy (IgH) chain in approximately 80% of follicular lymphomas; BCL2, BCL6, or MYC with IgH in approximately 50% of diffuse large B-cell lymphomas (DLBCLs)(5); and MYC with one of several Ig loci in 80% or more of Burkitt lymphomas.  The IgH locus is more prone to translocations because this locus undergoes two DNA recombination events, V(D)J recombination and class switching. Both these recombination events are critical for B cell development and function, but aberrant rearrangements lead to chromosomal translocations and cancer.  The importance of class switching in chromosomal translocations is further confirmed by the lack of c-myc/IgH translocations in activation induced cytidine deaminase (AID) deficient mice, AID being the enzyme responsible for initiating class switching. The precise molecular mechanism underlying chromosomal translocations remains largely unknown but it is clear that the key is aberrant repair of DNA breaks. Although significant advances have been made in understanding the mechanism of DNA repair but several major issues remain unresolved. Our lab wants to focus on studying the molecular mechanism of DNA repair in mammalian cells.

DNA damage and H2AX: One of the earliest events in the double stranded DNA break (DSB) response is the activation of the phosphatidylinositol-3 kinase-like family of kinases that include DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR). It is noteworthy that in mantle cell lymphoma defective DNA repair is caused by deletions of the ATM gene. The first target of these kinases is the histone H2A variant, H2AX (Fig.1). The phosphorylated form of H2AX (g-H2AX) has a role in repair, replication, recombination of DNA and regulating cell cycle. The large domains of g-H2AX generated at each DSB can be visualized by immunostaining as nuclear foci. The g-H2AX foci bind and retain an array of cell cycle and DNA repair factors (cohesins, MDC1, Mre11, BRCA1, 53BP1 etc.) at the break site.  Deletion of H2AX in mice results in aberrant repair culminating in oncogenic translocations and widespread tumors. Recent cytological studies in mouse B cells suggest that g-H2AX stabilizes the broken DNA ends during class switching giving the repair machinery sufficient time to make the appropriate joints.    It appears to regulate the kinetics of repair ensuring fidelity over rapid joining of random broken ends. Notably, H2AX maps to a cytogenetic region frequently altered in human cancers, possibly implicating similar functions in man.  Therefore the formation and removal of g-H2AX would be of utmost importance in the DNA repair process.  Although the kinases and stimuli involved in g-H2AX formation have been intensely investigated but how g-H2AX is eliminated in mammalian cells and what are the consequences of having constitutively phosphorylated H2AX remains unanswered.  The two possible mechanisms of g-H2AX removal from DNA damage sites are by histone exchange and/or by dephosphorylation by protein phosphatases. We investigated the role of phosphatases in the downregulation of g-H2AX.

g-H2AX and phosphatases: We found protein phosphatase 2A (PP2A) is involved in the removal of  g-H2AX foci in mammalian cells. PP2A is a major Ser/Thr phosphatase that regulates a variety of cellular processes, including signal transduction, apoptosis, DNA replication, cell cycle progression. The catalytic subunit [PP2A(C)] and a ‘constant’ regulatory subunit (A) associates with a ‘variable’ regulatory subunit (B) that is responsible for substrate specificity. We have shown that when PP2A(C) is inhibited or silenced by RNA interference, there is increase in cellular levels of g-H2AX with DNA damage and the g-H2AX foci persist relative to control cells. Impairing PP2A(C) by silencing or inhibitors affects multiple processes and we cannot determine the effect of PP2A on any isolated cellular pathway. One way to address this issue is to identify the B subunit responsible for PP2A activity on a particular substrate, and monitor PP2A function in the absence of the specific B subunit. Therefore we need to identify which B subunit targets the PP2A holoenzyme to g-H2AX.

Work in progress: Identify regulatory subunit (s) responsible for directing PP2A(C) to g-H2AX in cells.
Variable B subunits are classified into four families with each family having several members. In collaboration with the Hahn lab (Dana-Farber) we are making specific B subunit deficient cell lines by stable expression of small hairpins (sh) RNAs targeting the B subunit transcripts. We will then induce DNA damage and study levels of g-H2AX in these cells.  Due to the lack of antibodies against all these B subunits we are co-expressing tagged versions of the B subunits to investigate cellular localization in the context of DNA damage. A combination of these experiments will allow us to identify the B subunit targeting PP2A(C) to H2AX. Theoritically, in the absence of this B subunit the cell will specifically lack PP2A-mediated removal of g-H2AX. Assaying DNA repair efficiency and cell cycle progression in these B subunit deficient cells will elucidate the role of both PP2A and g-H2AX in these cellular processes.

Future Studies:

 Investigate the role of other PP2A-like phosphatases in the regulation of g-H2AX and DNA repair.
We have identified a yeast phosphatase (Pph3) that shares significant homology with PP2A(C) and dephosphorylates g-H2AX in a trimeric complex, HTP-C (histone H2A phosphatase complex). Both phosphatases efficiently dephosphorylate g-H2AX in vitro, but in cells there are key mechanistic differences between them.  Pph3 dephosphorylates g-H2AX only after it has been removed from chromatin whereas PP2A(C) colocalizes with DNA damage foci and is more likely to work in situ i.e. on the chromatin-bound g-H2AX.   Deletion of Pph3 has no effect on the kinetics of DNA repair and the rate of g-H2AX loss at a DSB.  The Pph3 mutants show a constitutive increase in g-H2AX levels even in the absence of an exogeneous DNA damaging agent. In PP2A-silenced cells DNA repair is inefficient and cells are hypersensitive to DNA damage. However these cells show no increase in constitutive levels of g-H2AX. Based on these results we hypothesize that g-H2AX is regulated by two types of phosphatases: One is a ‘constitutive’ phosphatase (e.g. Pph3 in yeast) that maintains the basal level of g-H2AX in the absence of a DSB response and the other an ‘inducible’ phosphatase (e.g. PP2A in mammalian cells) that specifically reduces elevated levels of g-H2AX during a DSB response. Our lab wants to investigate whether there are any phosphatases in mammalian cells that constitutively dephosphorylate g-H2AX.

Mammalian cells have two other phosphatases, PP4 and PP6, classified as PP2A-like based on homology to PP2A(C)(17).  PP4(C) is the closest human homolog of the yeast phosphatase Pph3.  PP4(C) forms several multimeric complexes with proteins with some homology to the subunits of the yeast phosphatase complex HTP-C. The cellular function and location of the different PP4-complexes is not known. Our preliminary experiments suggest that like Pph3, PP4(C) constitutively regulates g-H2AX. However, which PP4 complex is the functional analog of HTP-C remains to be investigated.  We have successfully designed and validated siRNAs and generated antibodies against all the subunits of different PP4 complexes in collaboration with Xavier Xu (Beijing University, China).  Using the biochemical and cytological techniques we standardized in our previous study with PP2A, we will identify the PP4 complex responsible for regulation of g-H2AX levels.  We will also study whether any of the PP4 complexes have a role in DNA repair and regulation of cell cycle. It is noteworthy that unlike kinases the role of phosphatases in DNA repair has not been well studied. Therefore a systematic investigation of phosphatases in DNA repair would be an important contribution to this field.

 Study the changes in chromatin structure accompanying a DSB

A DSB triggers a chain of events including the rapid formation of g-H2AX and recruitment of repair proteins at or near the DSB.  In mammalian cells these events have been defined primarily at the resolution of light microscopes. A partial molecular characterization of the chromatin changes that accompany a DSB has been done in yeast.  However in mammalian cells the limited resolution of light microscopy precludes a complete molecular-level picture of the events following a DNA break. A molecular insight into events in the vicinity of a DSB is the key to understanding the cause of chromosomal translocations.  We plan to use two different inducible DSB-generating systems to describe in detail the spatial and temporal regulation of DNA repair factors and chromatin around a DSB. Recombination activating gene (RAG) induced DSBs in murine pre-B cell lines and I-SceI induced DSB in human cell lines.  RAG-induced DSBs are unique and occur only in lymphocytes. I-SceI induces a ‘classical’ DSB, mimicking the breaks that occur during DNA replication or by ionizing radiation. These two systems complement each other and together will give us a comprehensive molecular description of DSB repair.
              In developing lymphocytes V(D)J recombination is initiated by RAG-induced DSBs at recombination signal sequences (RSS).  RAG-mediated cleavage occurs only in the G1-phase of the cell cycle and does not happen in dividing cells.  Abelson virus (V-abl) transformed pre-B cells are rapidly proliferating cells and have limited RAG activity. However treatment with abl kinase inhibitor, gleevac, leads to a synchronized block in G1 to S transition and rapid induction of RAGs followed by RAG-induced DSBs.  I-SceI is a rare-cutting yeast mitochondrial endonuclease that recognizes an 18-bp sequence with little degeneracy. I-SceI can therefore be used to introduce a DSB into a defined site in a mammalian chromosome. We have 293 (human embryonic kidney) cells with a single integrated copy of the I-SceI site, and expression of I-SceI in these cells leads to the formation of a DSB at this site.   Using chromatin immunoprecipitation we will study the kinetics and levels of recruitment of DNA repair proteins to the RAG-induced and I-SceI-induced DSB. Getting a detailed molecular image of DNA repair and elucidating the role of chromatin in this process will be of paramount importance in cancer biology.

Investigate role of microRNAs in repair of DSBs

MicroRNAs (miRs) are small (~22 bp) non-coding RNAs that regulate post-transcriptional gene expression by blocking translation of target mRNAs or by accelerating their degradation.  Since at least 30% of human genes are targeted by miRs it is safe to assume that miRs fine-regulate a diverse array of biological processes.  However, there have been no reports regarding their role in DNA repair pathways. There are two major DSB repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ). The nature of the DSB, and the stage of the cell cycle in which the break is induced determines the usage of DSB repair pathways. Rate and efficiency of repair of DSBs in resting vs. proliferating cells or in undifferentiated vs. terminally differentiated cells is different. One of the reasons for this disparity is differential expression of DNA repair and cell cycle regulatory factors.  We hypothesize that miRs regulate the cellular levels of DNA repair proteins, thus fine tuning the cellular response to a DSB.       
Using microarrays we have analyzed the expression profile of miRs in different sets of dividing and undifferentiated vs resting and differentiated cells. We have identified miRs that are differentially expressed in these distinct sets of cells. Different computational methods show that some of these miRs target DNA repair factors, and cell cycle regulatory proteins. We can modulate the expression of specific miRs using antagomirs and/or mimics and investigate whether these miRs, individually or in combination, affect the rate and capacity of DNA repair and cell cycle progression.  Identifying a role for miRs in DNA repair would potentially open up a new avenue of research in the DNA repair field.