The principal focus of our research is on understanding functions of poly(ADP-ribose)
polymerase 1 (PARP1) and the mechanisms of chromatin regulation by protein-poly(ADP-ribosyl)ation.
We have recently demonstrated that poly(ADP-ribose) polymerase 1 (PARP1) and poly(ADP-ribose)
glycohydrolase (PARG) perform a number of vital previously unrecognized functions
in a number of cellular processes (Tulin et al. 2002; Tulin et al. 2006). Distributed
evenly along chromatin (Figure 1AB) and enriched in nucleoli (Figure 1AB), PARP1 is
responsible for rapid local chromatin decondensation (loosening) (Figure 1), which
is required for transcriptional activation of many genes within specific chromatin
blocks (Tulin & Spradling 2003). Poly(ADP-ribosyl)ation is also involved in heterochromatin
formation and the initiation and maintenance of nucleoli (Figure 1D).
Tulin A, Stewart D, and Spradling AC (2002) The Drosophila heterochromatic gene encoding
poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during
development. Genes Dev. 16:2108-2119. PubMed Tulin A and Spradling A (2003) Chromatin loosening by poly(ADP)-ribose polymerase (PARP)
at Drosophila puff loci. Science 299:560-562.PubMed Tulin A, Chinenov Y, and Spradling A (2003) Regulation of chromatin structure and gene activity
by poly (ADP-ribose) polymerases. Curr. Top. Dev. Biol. 56:55-83. John Hopkins University Tulin A, Naumova N, Menon A, and Spradling A (2006) The Drosophila Poly(ADP-ribose) Glycohydrolase
(Parg) protein mediates chromatin structure and Sir2-dependent silencing. Genetics
H2Av Histone Controls PARP1 Protein Activation in Chromatin
We have successfully identified novel chromatin-associated PARP partners by implementing
a Tandem Affinity Purification (TAP) strategy together with sucrose gradient purification
and liquid chromatography-tandem mass spectrometry (LC-M.S./M.S.). Candidate interactors
arising from this approach were analyzed functionally for their influence on PARP,
using standard genetic approaches combined with immunostaining and confocal microscopy.
Based on this work, the variant histone H2Av (an H2Az, H2Ax homologue in Drosophila) has been identified as a protein that promotes targeting of PARP1 to chromatin.
Ser137-phosphorylated H2Av has been shown to co-localize with foci of local PARP activation in vivo (Meyer-Ficca et al., 2005). Our findings establish the importance of H2Av-PARP1 interaction
in terms of associating these proteins with chromatin. We propose the following model
for PARP1 binding to chromatin (Figure 2; Kotova et al., 2011): (1) H2Av establishes specific distinct domains in chromatin. (2) PARP interacts
with H2Av-positive chromatin. (3) Binding of PARP with chromatin is dynamic and H2Av-dependent.
(4) PARP1 enzymatic activity is neither required for its initial embedding in chromatin,
nor for its maintenance there. (5) Disruption of the interaction between PARP1 and
H2Av by phosphorylation of H2Av or mutation in H2Av phosphorylation domain leads to
spontaneous PARP1 activation.We focus our near-term efforts on validating the mechanisms of PARP1 targeting to
specific chromosomal domains using an extended in vivo and in vitro analysis of PARP1-H2Av
co-regulation, supported by mapping the subdomains of H2Av involved in these interactions.
Pinnola AD, Naumova N, Shah M, and Tulin AV(2007) Nucleosomal core histones mediate dynamic regulation of PARP1 protein binding
to chromatin and induction of PARP1 enzymatic activity. J. Biol. Chem. 282: 32511-32519.PubMed Kotova E, Jarnik M, and Tulin AV (2009) Poly (ADP-ribose) Polymerase 1 is required for protein localization to Cajal
body. PLoS Genetics. 5(2): e1000387. PubMed Kotova E, Jarnik M, and Tulin AV (2010) Uncoupling of the trans-activation and trans-repression functions of PARP1
protein. Proc. Natl. Acad. Sci. USA. 107(14): 6406-6411.PubMed Kotova E, Lodhi N, Jarnik M, Pinnola AD, Ji Y, andTulin AV (2011) Drosophila histone H2A variant (H2Av) controls Poly(ADP-Ribose) Polymerase
1 (PARP1) activation in chromatin. Proc. Natl. Acad. Sci. USA. 108(15): 6205-6210. PubMed Thomas CJ, Kotova E, Andrake M, Adolf-Bryfogle J, Glaser R, Regnard C, and Tulin AV(2014) Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation
via poly-ADP-ribosylation. Molecular Cell. 53: 831–842. PubMed
Poly(ADP-Ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates gene
Within the short span of the cell cycle, Poly(ADP-ribose) (pADPr) can be rapidly produced
by Poly(ADP-ribose) Polymerases (PARPs) and degraded just as rapidly by Poly(ADP-ribose)Glycohydrolases
(PARGs). The biological significance of pADPr oscillations in a living cell in steady
state conditions remains unclear. Recently, we identified heterogeneous nuclear RNP-binding
proteins (Hrps) as preferential PARP targets, which mediate PARP1 regulation of gene
expression during development (Figure 3; Ji & Tulin 2009). Here, we focus on investigating how changes in poly(ADP-ribosyl)ation of Hrp38
regulate tissue specification and planar polarity during oogenesis in Drosophila.
Specifically, turnover of poly(ADP-ribose) regulates Drosophila germ-line stem cell
maintenance and oocyte positioning by modifying Hrp38 protein function. Hrp38 binds
to the 5’UTR of DE-cadherin to promote its translation. Furthermore, pADPr binding
to Hrp38 in germ-line stem cell progeny disrupts the interaction of Hrp38 with the
5’UTR of DE-cadherin mRNA, thereby reducing DE-cadherin translation. Blocking the
translation of DE-cadherin leads to its depletion, which, in turn, causes differentiation
of progenitor cells. In agreement with these results, we found that defects in either
pADPr catabolism or Hrp38 function cause a decreased expression of DE-cadherin, which
leads to a loss of germ-line stem cells and mislocalization of oocytes in the ovary.
Taken together, our findings suggest that Hrp38 and its poly(ADP-ribosyl)ation regulate
DE-cadherin expression, and by doing so, control germ-line stem cell maintenance and
oocyte localization on the level of translation. Since PARP utilizes cellular NAD
to produce pADPr and its activity ultimately depends on the energy status of an organism,
we establish the chain of events that connect extrinsic conditions with the mode of
development and functioning of an organism.
Ji Y and Tulin AV(2009) Poly(ADP-ribosyl)ation of Heterogeneous Nuclear Ribonucleoproteins Modulates
Splicing. Nucl. Acids Research. 37: 3501-3513.PubMed Ji Y andTulin AV(2010) The roles of PARP1 in gene control and cell differentiation. Review for Curr.
Opin. Genet. Dev. 20(5):512-518. PubMed Ji Y and Tulin AV(2012) Poly(ADP-ribose) controls DE-cadherin-dependent stem cell maintenance and oocyte
localization. Nat Communications. 3:760. doi:10.1038/ncomms1759. PubMed Ji Y and Tulin AV(2013) Post-transcriptional regulation by Poly(ADP-ribosyl)ation. Int J Mol Sci. 14:
16168-16183. PMC Ji Y and Tulin AV(2013) Alternative Splicing Regulation by Poly(ADP-ribosyl)ation. Nova Science Publishers.
New Development in Alternative Splicing Research. pp. 159-170. Ji Y, Jarnik M, and Tulin AV(2013) Poly(ADP-ribose) Glycohydrolase and Poly(ADP-ribose)-interacting Protein Hrp38
Regulate Pattern Formation during Drosophila Eye Development. Gene. 526: 187–194. PubMed Ji Y, Thomas C,Tulin N, Lodhi N, Boamah E, Kolenko V, and Tulin AV (2016) Charon mediates IMD-driven PARP-1
dependent immune responses in Drosophila. Journal of Immunology. 197: 2382-2389. PMC Ji Y and Tulin AV(2016) Poly(ADP-ribosyl)ation of hnRNP A1 protein controls translational repression
in Drosophila. Molecular and Cellular Biology. 36(19): 2476-2486.MBC
Poly(ADP-Ribose) Polymerase 1 Regulates Nucleolar Functions and Translation
Poly(ADP-ribose)polymerase 1 (PARP1) is a nuclear protein that utilizes NAD to synthesize
poly(ADP)ribose (pADPr), resulting in both automodification and the modification of
acceptor proteins. Substantial amounts of nuclear PARP1 and pADPr (up to 50 %) (Figure
1) are localized in the nucleolus, a subnuclear organelle where ribosomal biogenesis
and maturation occur (Tulin et al., 2002, Tulin et al., 2006). At present, the functional
significance of PARP1 protein inside the nucleolus remains unclear.In this project, we investigate the roles that PARP1, pADPr, and PARP1-interacting
proteins play in the maintenance of nucleolus structure and functions. Our analysis shows that PARP1 interacts with a select group of nucleolar proteins.
Moreover, PARP1 is required for targeting these proteins to the proximity of pre-rRNA;
hence, PARP1 controls pre-rRNA processing, post-transcriptional modification and pre-ribosome
assembly. Importantly, we have observed that disruption of PARP1 enzymatic activity
causes nucleolus disintegration and aberrant localization of nucleolar-specific proteins.
Based on these findings, we propose a model that explains how PARP1 activity impacts
nucleolar functions and, consequently, ribosomal biogenesis.
During our preliminary research, we have identified a subset of ribosomal proteins
that bind preferentially to PARP1 and potential PARP1 targets, and, therefore, may
influence PARP1 regulation of nucleolar functions and translation during development
and stress response (Pinnola et al., 2007). Previous studies by other research groups
have confirmed strong PARP1 interaction with ribosomal protein L22 (rpl22) and ribosomal
protein L23 (rpl23)in vivo(Koyama et al., 1999). Subsequently, rpl22 protein was also suggested to control
global IRS-dependent translational initiation (Dr. David Wiest and Dr. Randy Strich,
personal communication). We are currently investigating PARP1 regulation of translation
and nucleolar integrity. We began by performing a functional study of the genetic
and biochemical interaction of PARP1 with rpl22 and RPL23. In addition, we plan to
investigate the mechanisms by which PARP1 regulates nucleolar functions and translation.
Boamah EK, Kotova E, Garabedian M, Jarnik M, and Tulin AV (2012) Poly(ADP-ribose) Polymerase 1 (PARP-1) regulates ribosomal biogenesis in Drosophila
nucleoli. PLoS Genetics. 8(1):e1002442.PubMed
PARP1 and pADPr are Epigenetic Marks in Mitotic Chromatin
The goal of this project is to determine how different kinds of new epigenetic marks
are distributed and stabilized specifically in mitotic chromatin. While many transcription
factors, co-regulators, and histone modifications exist in the interphase chromatin
and are important for gene regulation, those which occur in mitotic chromatin can
clearly confer nonmutational, epigenetic inheritance. Histone modifications are broadly
associated with either active or inactive genes, but do not set or re-set precise
gene expression states. Such states can be set by transcription factors and co-regulators.
However, most those that have been investigated, do not occupy mitotic chromatin and
hence cannot function as epigenetic marks. We have recently discovered that Poly(ADP-ribose)
Polymerase 1 (PARP1), which loosens local chromatin, along with poly(ADP ribose) (pADPr)
itself, are distinct from many other factors in that they stably occupy mitotic chromatin.
Given the ability of these factors to remain bound to chromatin through mitosis and
their ability to establish a transcriptionally competent state, we propose that PARP1
and pADPr, serve as a new class of epigenetic marks. We are examining the role of
poly-ADP-ribosylation in epigenetic regulation. Specifically, we plan to identify
sites of Poly(ADP-ribose) Polymerase 1 (PARP1) binding to mitotic and interphase chromatin.
We will also purify proteins associated with mitotic chromatin to uncover new epigenetic
regulatory factors. Our focus on mitotic events and new classes of epigenetic marks
provide novel directions for the field. Our work will reveal a mechanistic basis of
epigenetic inheritance and hence functional targets for developing therapeutic drugs
to ultimately treat epigenetics-based human diseases.
Lohdi N andTulin AV(2011) PARP1 Genomics: Chromatin Immunoprecipitation Approach using Anti-PARP1 Antibody
(ChIP and ChIP-seq). Methods Mol Biol. 780: 191-208. Lodhi N, Ji. Y, and Tulin AV(2016) Mitotic bookmarking: maintaining post-mitotic reprogramming of transcription
reactivation. Curr Mol Biol Rep. 2:10-16. Lodhi N, Kossenkov A, and Tulin AV (2014) Bookmarking promoters in mitotic chromatin: Poly(ADP-ribose)Polymerase-1 as
an epigenetic mark. Nucleic Acids Res. 42(11):7028-7038.PubMed
Poly(ADP-Ribosyl)ating Enzymes in Aging Control
In a eukaryotic organism, the progression of aging is associated with molecular changes
that regulate the transcriptional activation of age-related genes, the capacity for
DNA repair, programmed cell death, and a number of other vital processes. Accelerated
by environmental stress and damage accumulation, such changes ultimately lead to an
exponential increase in frailty and morbidity, and ultimately control the lifespan
of an organism. Poly(ADP-ribosyl)ation of proteins has long been known as a posttranslational
modification that is coupled to DNA repair and apoptosis triggering. Recently, the
crucial role of PARPs in a number of developmentally regulated processes, including
chromatin remodeling, transcriptional regulation, and telomere elongation has also
We plan to investigate how the regulation of a single pathway, the turnover of poly(ADP-ribose)
(pADPr), achieves the linkage between transcriptional regulation of specific genes
on the one hand and organismic aging and longevity on the other. Our preliminary results show that PARP1 activity is significantly elevated in aging
Drosophila. A considerable extension of the lifespan was observed for a fly strain
that has only one copy of the PARP1 gene when compared with wild type flies that carry
two copies of this gene. We have also demonstrated that PARG/PARP1 controls the activation
of a number of age specific genes. Knockdown of these genes leads to a pronounced
extension of life, whereas ectopic expression of these genes in young flies causes
premature aging and death. Additionally, we found that degree of poly(ADP-ribosyla)ation
of Lamin C--a protein involved in transcriptional silencing--is increased in old flies.
These findings allow us to propose that PARP1 protein is a principal regulator of
the aging program in Drosophila that controls aging program via regulation of transcription of age associated genes.
Publications in preparation.
New Approach to Identifying Poly(ADP-Ribose) Polymerase Inhibitors
During the past few years, Poly(ADP-ribose)Polymerase (PARP) proteins have become
a popular target for anti-cancer treatment. Many PARP inhibitors have been generated
and tested by the pharmacological industry. However, most of these inhibitors were
designed to disrupt the DNA-dependent PARP1 protein activation pathway, based on competition
with NAD for a binding site on the PARP molecule.This limitation resulted in discovery
of nucleotide-like PARP1-inhibitors that may target not only PARPs, but also other
enzymatic pathways involving NAD and nucleotides as co-factors. We are exploring a
strategy for the identification of PARP inhibitors that target a different pathway,
histone-dependent PARP1 activation. In addition to identification of NAD competitors
in a small molecules collection, this approach allows the discovery of novel classes
of PARP inhibitors that only disrupt histone-based steps of PARP1 activation and therefore
are more specific and more effective. This project will result in development of novel
small molecule inhibitors, which in future could be applied to anti-cancer treatment.
Kotova E, Pinnola AD, andTulin AV (2011) Small-molecule Collection and High-throughput Colorimetric Assay to Identify
PARP-1 Inhibitors. Methods Mol Biol. 780: 491-516 PubMed Tulin A(2011) Re-evaluating PARP1 inhibitor in cancer. Nat Biotechnol. 29(12):1078-9.Nature Biochemistry Kirsanov KI, Kotova E, Markov P, Golovine K, Lesovaya EA, Kolenko V, Yakubovskaya
MG, Tulin AV (2014) Minor grove binding ligands disrupt PARP-1 activation pathways. Oncotarget.
5: 428-437.PMC Thomas C, Ji Y, Kotova E, Lodhi N, Pinnola AD, Golovine K, Makhov P, Pechenkina K,
Kolenko V, Tulin AV (2016) New generation non-NAD-like PARP-1 inhibitors effectively eliminate drug resistant
tumors in vivo. EBioMedicine. 13 (2016) 90–98. Science Direct
Our long-term plan is to use the Drosophila system of poly(ADP-ribose) metabolism
to understand how a cell can be programmed to undergo quick, local and reversible
chromatin reprogramming and how this can be connected to the induction of local gene
activity. Eventually, genes located within repressed chromatin might be exposed and
made susceptible to re-programming by artificially activating nearby chromosomal PARP
molecules. Understanding of how PARP is activated within normal, undamaged chromatin
will advance our knowledge of developmental gene regulation and facilitate the development
of methods to experimentally re-program genes.
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