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Understanding Regulation of Macrophage Inflammatory
Response by Histone H3 Lysine 56 Acetylation
A project proposal submitted to
Department of Biotechnology,
Ministry of Science and Technology,
Govt. of India,
New Delhi-110 003
Dr. Devyani Haldar, Principal Investigator
Dr. Kishore Parsa, Co-investigator
Institute of Life Sciences
University of Hyderabad Campus
Gachibowli, Hyderabad 500046
Andhra Pradesh
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PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND
DEVELOPMENT, PROGRAMME SUPPORT
(To be filled by the applicant)
PART I: GENERAL INFORMATION
1. Name of the Institute/University/Organization submitting the Project Proposal: Institute of
Life Sciences
2. State: Andhra Pradesh
3. Status of the Institute: Public-Private Partnership Institute (recognized as SIRO)
4. Name and designation of the Executive Authority of the Institute/University forwarding the
application: Prof. Javed Iqbal, Director, Institute of Life Sciences
5. Project Title: Understanding regulation of macrophage inflammatory response by
Histone H3 lysine 56 acetylation
6. Category of the Project (Please tick): R&D
7. Specific Area: Medical Sciences and allied areas – Medical Biotechnology (infectious
disease).
8. Duration : 3 years 0 months
9. Total Cost (Rs.): 97 lakhs Rs. (94 lakhs + 3 lakhs overhead)
10. Is the project Single Institutional or Multiple-Institutional (S/M): S
11. If the project is multi-institutional, please furnish the following : N/A
Name of Project Coordinator: ...............................................................................
Affiliation: ...............................................................................................................
Address:
12. Scope of application indicating anticipated product and processes
This study would result in discovery of novel epigenetic regulation of inflammatory
response and also likely to contribute towards our understanding of molecular
mechanisms underlying the regulation of macrophage inflammatory response. This new
knowledge may provide a basis for development of novel therapeutic approaches for
several diseases which are associated with dysregulated inflammatory responses. .
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13. Project Summary (Not to exceed one page. Please use separate sheet).
Inflammatory response principally mediated by macrophages, is regulated by a sophisticated
regulatory network at signal-specific and gene-specific levels. Recent studies highlighted the crucial
role of histone modifications, including histone acetylation in regulation of inflammatory gene
expression; however our understanding about such regulatory mechanisms is still primitive and
warrants further investigation. Thus, in this study, we propose to undertake a comprehensive
investigation of global alterations in histone modifications in response to inflammatory stimuli by
western blotting using an array of commercially available antibodies. Further, in our preliminary
analysis, we observed that histone H3 lysine 56 acetylation (H3K56ac) is globally enhanced in time
dependent manner upon incubation of macrophages with LPS. In her previous work, PI has identified
that histone H3 is acetylated at lysine 56 residue and carried out ChIP on chip analysis which revealed
enrichment of H3K56ac on several genes associated with inflammation including TLR4, receptor for
bacterial endotoxin, LPS [1]. Thus, in this study, we seek to test the hypothesis that alterations in
histone H3K56 acetylation in response to inflammatory stimuli regulate macrophage inflammatory
response. Towards this objective, we first propose to systematically study the changes in the levels of
H3K56ac in response to LPS both globally and locally at the promoters of specific genes that we
identified from the previous ChIP on chip experiment (Figure 1). Second, we wish to examine the
association of this modification with the expression of inflammatory genes. Moreover, we aim to
identify the key signaling players including histone acetyl transferases such as CBP and Sirtuins
(histone deacetylases) involved in regulation of the H3K56ac in LPS exposed macrophages. Finally,
using a non-acetylatable H3K56 mimic, H3K56R, we intend to study the association of H3K56ac
with macrophage inflammatory response. We believe that this study will enhance our current
understanding about the epigenetic control of macrophage inflammation at the gene specific level and
that information gathered from this study will likely to permit devising novel therapeutic strategies
against diseases linked to dysregulated inflammatory responses.
Figure 1: Schematic representation of the proposed objectives of the study
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PART II: PARTICULARS OF INVESTIGATORS
Principal Investigator:
14. Name: Dr. Devyani Haldar
Date of Birth: 7th
January, 1971 Sex (M/F): F
Designation: Principal Research Scientist
Department: Biology
Institute/University: Institute of Life Sciences
Address:.University of Hyderabad Campus, Gachibowli, Hyderabad,
PIN 500046
Telephone: 040 6657 1500 Fax: 040 6657 1581 E-mail: [email protected]
Number of research projects being handled at present: Two (Two projects will end next
year)
Co-Investigator
16. Name: Dr. Kishore Parsa
Date of Birth: 31st August, 1975 Sex (M/F): M
Designation: Senior Research Scientist
Department: Biology
Institute/University: Institute of Life Sciences
Address: University of Hyderabad Campus, Gachibowli, Hyderababad,
PIN 500046
Telephone: 040 6657 1500 Fax: 040 6657 1581
E-mail: [email protected]
Number of Research projects being handled at present: Four (Three projects will end next
year)
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PART III: TECHNICAL DETAILS OF PROJECT
16. Introduction
16.1 Origin of the proposal
This proposal originates from the results obtained in an earlier research project in PI
laboratory [1]. In that project, we discovered a novel epigenetic modification of histone H3
core domain, the H3 lysine 56 acetylation (H3K56ac) and studied its function in DNA
damage response. In this study, a ChIP-on-chip analysis was carried out to investigate the
genome-wide distribution of H3K56ac. This study detected very high level of H3K56
acetylation at the promoters of genes involved in inflammatory response such as TLR3,
TLR4, TLR7, IRAK-1, AKT1, STAT-1, IL-1α etc. indicating it could be involved in
regulation of inflammatory response.
16.2 Definition of the problem
a) Rationale: Inflammatory response, principally mediated by macrophages, is regulated by
a sophisticated regulatory network to carry out functions at signal-specific and gene-specific
levels [2]. Recent studies highlighted the crucial role of epigenetic modifications, including
DNA methylation and covalent histone modifications in regulation of inflammatory gene
expression [3, 4]. Specifically, acetylation of histones is associated with regulation of gene
expression in response to multiple inflammatory stimuli. For example, TNF-α stimulated
H3K9 acetylation is the prerequisite for induction of some NF-κB dependent inflammatory
genes [5]. Enhanced acetylation of H3K56 was reported to augment Bclaf1 expression
during T cell activation [6]. However our understanding about the contribution of histone
modifications to control of inflammation and in particular to the activation of macrophages is
very primitive and requires comprehensive investigation. In an earlier study where PI
examined H3K56 acetylation by ChIP-on-chip analysis, enrichment of H3K56 acetylation at
several inflammatory genes was observed suggesting potential involvement of this
modification in the regulation of inflammatory response. Consistently, in our preliminary
analysis we noted that global H3K56 acetylation levels were robustly enhanced in
macrophages induced by bacterial endotoxin (Lipopolysaccharide; LPS) suggesting a
potential involvement of H3K56 acetylation in the control of macrophage inflammatory
response. Thus, in this project, we specifically seek to investigate the involvement of histone
aceytlation and in particular H3K56 acetylation in regulating inflammatory stimuli mediated
macrophage inflammatory response.
Hypothesis Alterations in histone H3K56 acetylation in response to inflammatory stimuli
regulate macrophage inflammatory response
Key questions
1) Does levels of histone modifications change globally in response to inflammatory stimuli?
2) Does H3K56 acetylation levels alter at the promoters of genes involved in LPS mediated
TLR4 signaling pathway?
3) Does alterations in H3K56 acetylation levels at the promoters influence the inflammatory
gene expression?
4) How is H3K56 acetylation level at the promoters of inflammatory response genes
regulated?
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16.3 Current status of research and development in the subject
International and national status
Covalent modifications of histone such as acetylation, methylation and phosphorylation were
observed to alter in response to different inflammatory stimuli and these changes are linked
to changes in gene expression [3, 7]. For instance, the locus of TNF-α, a potent cytokine
which co-ordinates inflammatory responses via multiple ways, shows regions of focused
acetylation (H3 and H4) and methylation (H3 dimethyl and H3 trimethyl) imprints in
response to LPS [8]. Further it was reported that decreased H3 acetylation and H3S10
phosphorylation is associated with decreased recruitment of p65 to the Tnf promoter which
resulted in inhibition of TNF-α (but not IL-10) production during infection with Toxoplasma
gondii [9]. Alterations in positive histone marks, H4 acetylation and trimethylation of H3K4,
were observed at specific genes in LPS tolerant macrophages [10]. Interestingly, gene-
specific chromatin modifications were associated with transcriptional silencing of a class of
genes which are pro-inflammatory in nature and priming of another class of genes which
encode anti-microbial effectors thus explaining differential regulation of different classes of
genes during endotoxin tolerance [10]. Another study identified that a subset of LPS-
inducible genes undergo signal-dependent demethylation of trimethylated H3K27 as a
prerequisite for their induction. The demethylase 6B (KDM6B; also known as JMJD3) is
responsible for this activity, which is itself transcriptionally up-regulated after LPS
stimulation, and siRNA-mediated knockdown of KDM6B inhibited demethylation and the
induction of Bmp2 (bone morphogenetic protein 2) expression [11]. However, deletion of
Jmjd3 failed to impair the secretion of pro-inflammatory mediators in response to TLR
ligands or Listeria monocytogenes infection but suppressed the alternative activation of
macrophages in response to helminth infection or chitin administration [12, 13]. In another
study, it was elegantly shown that HDAC3 applies an epigenomic brake on the alternative
activation of macrophages [14]. Stimulation of macrophages with IL-4 markedly induced
H3K9 acetylation at specific genes and deletion of HDAC3 exaggerated this response
consistent with enhanced expression of alternative macrophage activation markers [14].
Further, deletion of HDAC3 exerted protective effects during Schistosoma mansoni egg
challenge, a model of Th2 cytokine-mediated disease limited by alternative activation of
macrophages [14]. SIRT proteins, which are nicotinamide adenine dinucleotide (NAD)-
dependent deacetylases of the class III HDAC family, have also been recently implicated in
the transcriptional control of inflammatory genes. SIRT1 deacetylates NF-κB p65
transactivation domain, which blocks p65-dependent gene induction that is independent of
DNA binding [15]. SIRT6 also inhibits NF-κB activity albeit through a different mechanism.
SIRT6 directly deacetylates H3K9 at the promoters of certain TNF-α induced NF-κB-
regulated genes [5]. Acetylated H3K9 is closely associated with transcriptional activation
across the genome, and SIRT6-mediated deacetylation of H3K9 represses both basal and
stimulus-dependent gene induction [5]. Moreover, a recent study has shown that Sirt1
inhibits T cell activation via H3K56 deacetylation at the promoter region to inhibit
transcription of Bclaf1 [6].
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16.4 The relevance and expected outcome of the proposed study
The above described studies clearly highlight the emerging role of histone modifications in
regulating macrophage inflammatory gene expression programmes however several key
questions still remain unanswered. Thus, this study will add to our basic understanding of the
involvement of histone acetylation, particularly addition of acetyl group to H3K56, in
regulating the process of macrophage mediated inflammatory response. Deciphering the
epigenomic basis of macrophage dependent responses will undoubtedly assist in devising
novel therapeutic strategies against various diseases such as autoimmune disorders, cancer,
diabetes, atherosclerosis etc. which are linked to dysregulated inflammatory responses. In
fact, recently a novel synthetic compound (I-BET) is developed and was demonstrated to
mimic acetylated histones and thereby disrupt chromatin complexes responsible for the
expression of key inflammatory genes in activated macrophages [16]. Consistently, I-BET
conferred protection against endotoxin induced lethality and bacterial sepsis [16] providing
the proof of concept.
16.5. Review of expertise available with proposed investigating group/institution in the
subject of the project
Dr. Devyani Haldar, currently leading a team of biologists at the Institute of Life Sciences
has over 11 years of research experience. She has considerable experience in area of
chromatin biology and has demonstrated skill sets for implementation of this project. Her
group is one of the first four groups to discover H3K56 acetylation in mammalian cells. This
project has originated from her earlier published work on H3K56ac [1]. She has two ongoing
DBT funded projects studying various aspects of biological functions of histone deacetylases
(Sirtuin family). Her research group in collaboration with co-PI Dr. Parsa’s lab has already
set up the experimental system required for this study (as shown in preliminary results figure
1).
Dr. Kishore Parsa is a senior research scientist leading a team of biologists at the Institute of
Life Sciences and has an overall 5 years of research experience. During this period he has
demonstrated the skill set required for implementation of the proposed project. He authored
28 articles including 21 relevant to the current research project demonstrating required
expertise to successfully execute the proposed project. Dr. Parsa is currently funded by DBT,
CSIR and DAE to study the molecular mechanisms of macrophage activation.
16.6. Patent details (domestic and international):
none
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16.7 Preliminary work done so far
To study the global alterations in histone modifications in response to different
inflammatory stimuli.
To investigate the alterations in the level of histone acetylation in response to inflammatory
stimulus, we have treated RAW 264.7 macrophages with 1 µg/ml of LPS for 6, 12 and 24
hrs. Histones were isolated and the levels of histone H3K56 acetylation and histone H4K16
acetylation were monitored by western blotting. LPS treatment resulted in a marginal
increase of global H3K56 acetylation levels at 6 h, however prolonged exposure of
macrophages robustly enhanced histone H3K56 acetylation levels. In contrast, un-stimulated
(resting) cells displayed high levels of H4K16ac and no further enhancement was observed
due to LPS stimulation (Figure 2).
Figure 2: The level of acetylation of histone H3K56 altered on LPS treatment in
macrophages
To determine if inflammatory pathway genes contain acetylation of H3K56 at their
promoters, we analyzed the data from an earlier ChIP-on-chip study carried out by PI to
check the genome-wide occupancy of H3K56ac [1]. This analysis showed that H3K56
acetylation was enriched at the promoters of several genes of inflammatory signaling
pathways. Figure 3 shows the identity and fold enrichment of selected inflammatory response
genes. As shown below several of the genes including the receptor for LPS, TLR4, and its
key downstream signaling players such as IRAK1, AKT1, STAT1, IL1β etc were observed to
contain enriched H3K56ac mark. Enrichment of H3K56ac mark at TLR4 was validated in our
previous study [1].
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Figure 3: ChIP-on-chip data showing enrichment of acetylation of histone H3K56 at the
promoters of inflammatory response genes
17. Specific objectives
The objectives of this study are:
1) To study the global alterations in histone modifications in response to different
inflammatory stimuli.
To investigate this, we will expose macrophages/monocytes to different concentrations of
LPS, IFN-γ, IL-4 and IL-13 for varied time points and the status of different histone
modifications mainly acetylation and methylation at specific residues will be analyzed by
western blotting. Determination of genome-wide distribution of histone H3K56 acetylation
in unstimulated and LPS treated macrophages will be carried out by ChIP-seq analysis.
Verifiable indicators of progress: Preliminary analysis has revealed enhanced acetylation of
H3K56 when macrophages were incubated with LPS. Determination of acetylation status of
other histone residues will clearly indicate the progression of this aim.
2) To examine the changes in H3K56 acetylation levels at specific inflammatory
response genes and determine its effect on their expression.
For this, we will stimulate macrophages with LPS for different time points and status of
H3K56 acetylation at selected genes such as TLR4, IRAK1, miR-155 etc. will be monitored
by ChIP-qPCR. In parallel, macrophages will be transfected with non-acetylatable H3K56R
mutant construct and the effect of suppressed H3K56 acetylation on the expression of
specific genes will be tested by western blotting.
Verifiable indicators of progress: Majority of the experiments proposed under this aim
require analysis by ChIP-qPCR, thus optimization of conditions for ChIP-qPCR will indicate
the progression of this specific objective.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
TLR4 TLR3 TLR7 AKT1 AKT3 IRAK1STAT1 IL1-α IL1-β
Fold
H3
K5
6ac
en
rich
me
nt 3
14 1
1
1
1 11
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3) To elucidate the functional role of H3K56 acetylation in regulation of
inflammatory response.
To determine the functional consequence of LPS-stimulated H3K56 acetylation, we will
over-express non-acetylatable H3K56R mutant in macrophages and will determine its effect
on their activation by monitoring selected inflammatory markers such as cytokines and
reactive intermediates by ELISA and fluorescent dyes, respectively. In addition, we will also
identify the enzymes that regulate LPS-induced H3K56 acetylation and will then assess the
role of such enzymes in macrophage activation as described above.
Verifiable indicators of progress: Over-expression of H3K56R mutant or knock down of
selected histone modifying enzymes and demonstration of altered global/local H3K56
acetylation signals undoubtedly signifies advancement of this aim.
18. Work plan
Specific aim 1: To study the global alterations in histone modifications in response to
different inflammatory stimuli.
18.1 Work plan (methodology/experimental design to accomplish the stated aim)
Epigenetic regulation of inflammatory response has become evident from several recent
reports [3, 7]. Along with other kinds of epigenetic phenomena, few studies have pointed out
the emerging role of histone modifications in regulation of inflammatory response [3, 7].
Although there are isolated reports of alteration of histone modifications in response to
inflammatory stimuli but there is no comprehensive study. Thus, here we propose to study the
alterations in the levels of histone modifications in response to different inflammatory
stimuli. To investigate the role of histone modifications in the control of inflammatory
response, we will expose RAW 246.7 macrophages to various concentrations of different
inflammatory stimuli such as LPS, IFN-γ, IL-4 and IL-13 for varied time points and the status
of different histone modifications mainly acetylation (H3K9ac, H3K56ac, H4K16) and
methylation (H3K4me, H3K9me, H3K27me) at specific residues will be analyzed by
isolating histones from un-stimulated and stimulated cells, separating them on SDS-PAGE
followed by western blotting using commercially available antibodies that detect specific
histone modifications. The status of these modifications in response to inflammatory stimuli
will also be checked in THP1 and U937 cells.
To further examine the genome-wide changes in H3K56 acetylation levels in response to
inflammatory stimuli, a ChIP-seq analysis will be carried out. In our preliminary study, we
have observed a significant increase in the global H3K56ac on LPS treatment; therefore, to
understand the role of this acetylation in LPS mediated macrophage inflammatory response,
we propose to carry out a ChIP-seq analysis. For this, a total of 1x108 RAW 264.7 cells will
be treated with for 1µg/ml LPS for 24 h and will be subjected to chromatin
immunoprecipitation (ChIP). ChIP will be performed essentially as described earlier [17, 18].
Briefly, formaldehyde crosslinked cells will be sonicated to obtain fragments of genomic
DNA ranging between 200 and 1000 bp. Sonicated chromatin will be immunoprecipitated
using anti-H3K56ac. Acetylated H3K56 associated chromatin will be deproteinized and used
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for ChIP-seq. The ChIP-Seq libraries will be prepared as per Illumina's instructions. Briefly,
ChIP sample DNA fragments will be blunted, phosphorylated, and ligated to library adapters
provided through Illumina. For input DNA preparation, 10 ng of starting material will be
used. Following ligation, size selection will be performed by gel electrophoresis by excising
DNA fragments at 200 ± 25 base pairs. Following gel purification, PCR amplification will be
performed. Amplified material will be run on the Agilent 2100 bioanalyzer using the DNA
1000 kit to ensure proper size selection, and will be
subsequently diluted to a concentration of 10 nM. These products will be sequenced on the
Illumina 1G Genome Analyzer at a concentration of 3–4 pM [18]. The data will be analyzed
by GLITR (GLobal Identifier of Target Regions) software [18]. The above experiments will
establish if specific histone modification levels are altered in response to specific
inflammatory conditions. The ChIP-seq analysis will aid in further confirming the enrichment
of H3K56 acetylation at the promoters of inflammatory genes and gain new insights into
acetylation status of these promoters in response to LPS stimulation.
18.2 Connectivity of the participating institutions and investigators ( in case of multi-
institutional projects only):
Not applicable
18.3 Alternate strategies (if the proposed experimental design or method does not work
what is the alternate strategy):
We have extensive experience in performing the proposed experiments and we do not
anticipate any major problems during the study [1, 19]. The above experiments to study
alterations in histone modification under inflammatory conditions will of course yield result.
Our preliminary data indicate towards this outcome. Our preliminary data (Figure 2) suggests
that H3K56ac is enhanced during LPS stimulation and we expect that methylation of H3
which is shown to polarize macrophage inflammatory response may be altered globally;
however if we observe that global histone acetylation is insensitive to inflammatory stimuli
then alterations in methylation status of H3 will be examined at specific genes. Further, the
effect of inflammatory stimuli on other epigenetic post-translational modifications such as
histone phosphorylation will be investigated. Although histone phosphorylation is less
studied than other modifications such as methylation and acetylation but emerging data
suggests that macrophage inflammatory response is also controlled by phospho modification
of H3 [20-22].
Specific aim 2: To examine the changes in H3K56 acetylation levels at specific
inflammatory response genes and determine its effect on their expression.
18.1 Work plan (methodology/experimental design to accomplish the stated aim)
In our earlier study, we have observed enrichment of H3K56 acetylation at the promoters of
several inflammatory response genes. However, it is not known whether this also happens in
macrophages and if H3K56 acetylation levels change when macrophages are activated by
LPS.
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Here we seek to investigate if H3K56 acetylation levels are altered at the promoters of genes
involved in LPS mediated TLR4 signaling pathway and if so, whether these alterations at the
promoters influence the expression of these genes. To test this we will perform ChIP with
anti H3K56 antibodies followed by qPCR of precipitated chromatin fragments. For this
experiment, RAW 264.7 cells will be treated with different concentrations of LPS for
different time points and used for chromatin immunoprecipitation. ChIP will be performed
essentially as described before [17]. Briefly, formaldehyde cross linked cells will be
sonicated to obtain fragments of genomic DNA ranging between 200 and 1000 bp. Sonicated
chromatin will be immunoprecipitated using anti-H3K56ac. Acetylated H3K56 associated
chromatin will be deproteinized and qPCR will be carried out with these ChIPed DNA for
genes of TLR4 signaling pathway such as TLR4, IRAK1, AKT, STAT1, IL1-beta etc. using
SYBR green chemistry. Following this, the expression of above genes at different time points
and concentrations of LPS will be examined by real time RT-PCR using SYBR green
chemistry. β-actin will be employed as the reference gene control.
To test if the H3K56ac levels directly influence the expression of genes of TLR4
inflammatory signaling pathway, a non-acetylatable mimic of histone H3, H3K56R and H3
wild-type will be over-expressed in macrophages and global H3K56ac will be monitored by
western blot. The point mutation of the H3 sequence will be created with arginine substituted
for lysine at the amino acid position 56 utilizing site directed mutageneis kit. RAW 264.7
cells will be transfected with H3K56R or H3 wild-type expressing construct by Amaxa
Nucleofector apparatus as we previously described [19]. Sixteen hours post transfection cells
will be exposed to LPS for different time points and subsequently expression of the genes
will be studied by qPCR.
18.2 Connectivity of the participating institutions and investigators (in case of multi-
institutional projects only):
Not applicable
18.3 Alternate strategies (if the proposed experimental design or method does not work
what is the alternate strategy):
With the above set of experiments, we anticipate to demonstrate that acetylation of H3K56 is
augmented locally at the promoters of selected genes in TLR4 pathway. Further, we also
predict that changes in H3K56ac status is linked to their expression. However, it is possible
that H3K56ac is unaltered at the promoters of any of the selected genes. We believe that this
situation may arise due to two scenarios. First, the minimal region (~150-200 bp) of the
promoter of specific gene selected for qPCR primer design may not harbor the enriched
H3K56ac mark. In such a case several primer pairs will be designed to scan the entire
promoter region of the selected gene. Second, H3K56ac is indeed not modified at any region
of the promoter of the selected gene. If it were to be this situation, then our analysis will be
guided by the data obtained in genome-wide ChIP-Seq analysis of H3K56ac in LPS-
stimulated macrophages (Specific objective 1).
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Specific aim 3: To elucidate the functional role of H3K56 acetylation in regulation of
inflammatory response.
18.1 Work plan (methodology/experimental design to accomplish the stated aim)
Building upon our preliminary data that H3K56 acetylation mark is enriched on inflammatory
genes (Figure 3) and global augmentation of H3K56ac levels in LPS exposed macrophages
(Figure 2), here we seek to characterize the functional relevance of H3K56ac in regulating
the activation of macrophages by LPS. Specifically we wish to study two major aspects. First,
we will examine and identify the signaling events that establish and erase the H3K56ac
imprints on promoters of inflammatory genes during macrophage activation. Second, we will
investigate the role of H3K56ac in the control of LPS induced macrophage inflammatory
responses.
Previously, PI has shown that basal acetylation levels of H3K56 is maintained by
orchestrated action of histone acetyl transferase CBP and histone deacetyltransferases SIRT2
and SIRT3 [1]. Other research groups have demonstrated that SIRT1, SIRT2 and SIRT6 are
also competent to remove H3K56 acetylation mark [23, 24]. Thus, to identify the enzymes
that modify H3K56ac levels in LPS exposed macrophages we will undertake a genetic
approach. For this, the expression levels of H3K56ac modifiers mentioned above will be
altered by nucleofecting RAW 264.7 macrophages with cDNAs or specific siRNAs or with
respective controls. Post 16-48 h of transfection, cells will be exposed to 1 µg/ml of LPS for
different time points and the global H3K56ac levels will be determined by western blotting
analysis. Additionally, to specifically examine the local changes in H3K56ac levels at
selected genes, transfected cells will be subjected to ChIP-qPCR assay as described above.
H3K56ac is linked to gene expression, thus, we will also assess the implication of the
selected histone modifying enzyme in the expression of inflammatory genes. For this, cDNA
or siRNA transfected cells will be exposed to 1 µg/ml of LPS for different time points. Un-
stimulated cells will serve as negative controls. Post-stimulation, cells will be lysed; total
RNA will be harvested using TRIzol reagent followed by phenol/chloroform extraction and
isopropyl alcohol precipitation. Subsequently, total RNA will be reverse transcribed and
analyzed by Real-Time PCR using SYBR green chemistry. β-actin will be employed as the
reference gene control. Engagement of TLR4 by its ligand LPS causes the activation of
diverse signaling pathways. Thus, to probe the identity of upstream signaling events that
control H3K56ac levels, before LPS stimulation we will pre-incubate cells with
pharmacological inhibitors of MAPKs (ERK1/2, p38, JNK), PI3K/Akt etc. which relay the
signal from the receptor to the target gene. Subsequently, global and local levels of H3K56ac
at specific genes will be monitored as described above. Data obtained using inhibitors will be
further confirmed by genetic approaches as described above.
Finally, we seek to investigate the specific role of H3K56ac in modulating macrophage
inflammatory responses. It is well established that histone modifications are linked to
biological response; however it is not always easy to establish the causative nature of a
modification and in many instances correlative evidence was provided. Proving causality of
the modification for ensuing biological response may involve delineating the contribution of
the histone modifying enzyme to the regulation of response, nevertheless this approach is
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mired by issues such as signaling redundancy, multiple substrates etc. Another way that
partially circumvents the above issues is to introduce dominant negative histone mutant to
compete out endogenous wild type histone and then assess the causative nature of the
particular modification. Both the approaches are reported in literature [6, 25] and in this aim
both the strategies will be explored. In the first approach, we will genetically alter the
expression of histone modifying enzymes either by over-expression or by siRNA mediated
RNAi as described above and then cells will be stimulated with 1 µg/ml of LPS for different
time points. Post stimulation, cell supernatants will be harvested and the secretion of
cytokines such as TNF-α, IL-12, IL-6 etc. will be analyzed by cytokine specific ELISA kits.
Further, we will also measure the levels of reactive oxygen species and nitric oxide by using
fluorescent dyes DCFDA and DAF, respectively. In the second approach, cells will be
transfected with wild type H3 or H3K56R dominant negative mutant, 16 h post
nucleofection, cells will be stimulated with LPS for different time points and a panel of
inflammatory readouts described above will be measured. In parallel, control experiments
will be performed to test whether H3K56ac levels are diminished globally and/ locally in
H3K56R transfected cells.
18.2 Connectivity of the participating institutions and investigators (in case of multi-
institutional projects only):
Not applicable
18.3 Alternate strategies (if the proposed experimental design or method does not work
what is the alternate strategy):
We demonstrated extensive experience in performing majority of the proposed experiments
and thus we do not expect any major potential pitfalls in the experimental methods [1, 19].
However as noted above proving the causality of the H3K56ac to modulation of macrophage
inflammatory responses may be a challenging task and we are addressing this question in two
ways as described above. It is possible that transient over-expression of non-acetylatable
H3K56R mimic does not apparently effect macrophage inflammatory response due to
masking of the mutant effect by un-transfected cells which does not express the mutant. In
such a case, a cell population which stably expresses the mutant H3 will be generated and
will be used for the proposed experiments. Alternatively, an inducible system to knockdown
endogenous H3 and over-express mutant H3 will be considered. It is possible to selectively
knockdown endogenous H3 sparing mutant H3 by targeting the 3’UTR region of the H3
transcript.
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References
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mediated acetylation of histone H3 lysine 56 functions in DNA damage response in
mammals. J Biol Chem 285, 28553-28564.
2. Medzhitov, R., and Horng, T. (2009). Transcriptional control of the inflammatory
response. Nat Rev Immunol 9, 692-703.
3. Bayarsaihan, D. Epigenetic mechanisms in inflammation. J Dent Res 90, 9-17.
4. Fuchs, J., Demidov, D., Houben, A., and Schubert, I. (2006). Chromosomal histone
modification patterns--from conservation to diversity. Trends Plant Sci 11, 199-208.
5. Kawahara, T.L., Michishita, E., Adler, A.S., Damian, M., Berber, E., Lin, M., McCord,
R.A., Ongaigui, K.C., Boxer, L.D., Chang, H.Y., and Chua, K.F. (2009). SIRT6 links
histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and
organismal life span. Cell 136, 62-74.
6. Kong, S., Kim, S.J., Sandal, B., Lee, S.M., Gao, B., Zhang, D.D., and Fang, D. The type
III histone deacetylase Sirt1 protein suppresses p300-mediated histone H3 lysine 56
acetylation at Bclaf1 promoter to inhibit T cell activation. J Biol Chem 286, 16967-
16975.
7. Takeuch, O., and Akira, S. Epigenetic control of macrophage polarization. Eur J Immunol
41, 2490-2493.
8. Sullivan, K.E., Reddy, A.B., Dietzmann, K., Suriano, A.R., Kocieda, V.P., Stewart, M.,
and Bhatia, M. (2007). Epigenetic regulation of tumor necrosis factor alpha. Mol Cell
Biol 27, 5147-5160.
9. Leng, J., Butcher, B.A., Egan, C.E., Abi Abdallah, D.S., and Denkers, E.Y. (2009).
Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered
macrophage activation. J Immunol 182, 489-497.
10. Foster, S.L., Hargreaves, D.C., and Medzhitov, R. (2007). Gene-specific control of
inflammation by TLR-induced chromatin modifications. Nature 447, 972-978.
11. De Santa, F., Totaro, M.G., Prosperini, E., Notarbartolo, S., Testa, G., and Natoli, G.
(2007). The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of
polycomb-mediated gene silencing. Cell 130, 1083-1094.
12. Satoh, T., Takeuchi, O., Vandenbon, A., Yasuda, K., Tanaka, Y., Kumagai, Y., Miyake,
T., Matsushita, K., Okazaki, T., Saitoh, T., Honma, K., Matsuyama, T., Yui, K.,
Tsujimura, T., Standley, D.M., Nakanishi, K., Nakai, K., and Akira, S. The Jmjd3-Irf4
axis regulates M2 macrophage polarization and host responses against helminth infection.
Nat Immunol 11, 936-944.
13. Ishii, M., Wen, H., Corsa, C.A., Liu, T., Coelho, A.L., Allen, R.M., Carson, W.F.t.,
Cavassani, K.A., Li, X., Lukacs, N.W., Hogaboam, C.M., Dou, Y., and Kunkel, S.L.
(2009). Epigenetic regulation of the alternatively activated macrophage phenotype. Blood
114, 3244-3254.
14. Mullican, S.E., Gaddis, C.A., Alenghat, T., Nair, M.G., Giacomin, P.R., Everett, L.J.,
Feng, D., Steger, D.J., Schug, J., Artis, D., and Lazar, M.A. Histone deacetylase 3 is an
epigenomic brake in macrophage alternative activation. Genes Dev 25, 2480-2488.
15. Yeung, F., Hoberg, J.E., Ramsey, C.S., Keller, M.D., Jones, D.R., Frye, R.A., and Mayo,
M.W. (2004). Modulation of NF-kappaB-dependent transcription and cell survival by the
SIRT1 deacetylase. EMBO J 23, 2369-2380.
16. Nicodeme, E., Jeffrey, K.L., Schaefer, U., Beinke, S., Dewell, S., Chung, C.W.,
Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kirilovsky, J., Rice, C.M.,
Lora, J.M., Prinjha, R.K., Lee, K., and Tarakhovsky, A. Suppression of inflammation by a
synthetic histone mimic. Nature 468, 1119-1123.
15 | P a g e
17. Kumar, P.P., Purbey, P.K., Ravi, D.S., Mitra, D., and Galande, S. (2005). Displacement
of SATB1-bound histone deacetylase 1 corepressor by the human immunodeficiency
virus type 1 transactivator induces expression of interleukin-2 and its receptor in T cells.
Mol Cell Biol 25, 1620-1633.
18. Tuteja, G., White, P., Schug, J., and Kaestner, K.H. (2009). Extracting transcription factor
targets from ChIP-Seq data. Nucleic Acids Res 37, e113.
19. Parsa, K.V., Ganesan, L.P., Rajaram, M.V., Gavrilin, M.A., Balagopal, A., Mohapatra,
N.P., Wewers, M.D., Schlesinger, L.S., Gunn, J.S., and Tridandapani, S. (2006).
Macrophage pro-inflammatory response to Francisella novicida infection is regulated by
SHIP. PLoS Pathog 2, e71.
20. Lucas, M., Zhang, X., Prasanna, V., and Mosser, D.M. (2005). ERK activation following
macrophage FcgammaR ligation leads to chromatin modifications at the IL-10 locus. J
Immunol 175, 469-477.
21. Yang, S.R., Valvo, S., Yao, H., Kode, A., Rajendrasozhan, S., Edirisinghe, I., Caito, S.,
Adenuga, D., Henry, R., Fromm, G., Maggirwar, S., Li, J.D., Bulger, M., and Rahman, I.
(2008). IKK alpha causes chromatin modification on pro-inflammatory genes by cigarette
smoke in mouse lung. Am J Respir Cell Mol Biol 38, 689-698.
22. Hasegawa, Y., Tomita, K., Watanabe, M., Yamasaki, A., Sano, H., Hitsuda, Y., and
Shimizu, E. (2005). Dexamethasone inhibits phosphorylation of histone H3 at serine 10.
Biochem Biophys Res Commun 336, 1049-1055.
23. Schwer, B., Schumacher, B., Lombard, D.B., Xiao, C., Kurtev, M.V., Gao, J., Schneider,
J.I., Chai, H., Bronson, R.T., Tsai, L.H., Deng, C.X., and Alt, F.W. Neural sirtuin 6
(Sirt6) ablation attenuates somatic growth and causes obesity. Proc Natl Acad Sci U S A
107, 21790-21794.
24. Das, C., Lucia, M.S., Hansen, K.C., and Tyler, J.K. (2009). CBP/p300-mediated
acetylation of histone H3 on lysine 56. Nature 459, 113-117.
25. Abbosh, P.H., Montgomery, J.S., Starkey, J.A., Novotny, M., Zuhowski, E.G., Egorin,
M.J., Moseman, A.P., Golas, A., Brannon, K.M., Balch, C., Huang, T.H., and Nephew,
K.P. (2006). Dominant-negative histone H3 lysine 27 mutant derepresses silenced tumor
suppressor genes and reverses the drug-resistant phenotype in cancer cells. Cancer Res
66, 5582-5591.
16 | P a g e
19. Timelines:
Period of study Activity
6 Months
Purchase of equipment and consumables etc.
Recruitment of manpower
Global analysis of histone modifications in response to
different inflammatory stimuli.
Site-directed mutagenesis to construct H3K56R mutant
12 Months Continued: Global analysis of histone modifications in
response to different inflammatory stimuli.
Optimization of ChIP-qPCR conditions
Genome-wide analysis of H3K56Ac in LPS stimulated
macrophages
18 Months Analysis of H3K56ac at specific genes by ChIP-qPCR
Determination of the effect of H3K56ac on the expression of
selected genes by Real-Time RT-PCR
24 Months Continued: Determination of the effect of H3K56ac on the
expression of selected genes by Real-Time PCR
Characterization of the role of selected HATs and HDACs in
the regulation of LPS-stimulated H3K56ac.
30 Months Continued: Characterization of the role of selected HATs
and HDACs in the regulation of LPS-stimulated H3K56ac.
Investigation of upstream signaling involved in LPS-induced
H3K56ac augmentation
36 Months Studying the role of selected HAT/HDACs in the regulation
of LPS-mediated inflammatory response
Determination of the effect of H3K56ac on the expression of
cytokines and oxidative burst by ELISA and fluorescent
dyes.
20. Name and address of 5 experts in the field
Sr. No. Name Designation Address
1.
Prof. Seyed E
Hasnain Professor
Kusuma School of Biological
Sciences
Indian Institute of Technology-Delhi,
Hauz Khas, New Delhi 110 016
India.
Email: [email protected]
2. Dipankar Chatterji Professor
Molecular Biophysics Unit, Indian
Institute of Science, Bangalore 560
012
Email: [email protected]
3.
Kanury Venkata
Subba Rao
Group
Leader
ICGEB Laboratories
ICGEB Campus
Aruna Asaf Ali Marg
110 067 New Delhi
Email: [email protected]
17 | P a g e
4.
Dr. Sharmistha
Banerjee
Reader
Department of Biochemistry
University of Hyderabad,
Gachibowli,
Hyderabad-46, India.
Ph:91-40-23134573
Email: [email protected]
5.
Dr. MS Reddy
Staff
Scientist
Center for DNA Fingerprinting and
Diagnostics [CDFD]
Bldg. 7, Gruhakalpa, 5-4-399 / B,
Nampally, Hyderabad 500 001
Email: [email protected]
18 | P a g e
PART IV: BUDGET PARTICULARS
A) Non-Recurring (e.g. equipments, accessories, etc.)
~Rs. in lakhs
S. No. Item Year 1 Year 2 Year 3 Total
Major Equipment
1 Real time PCR machine 17 - - 17
2 Bioruptor Standard sonication
device with accessories 11 - - 11
3 Vertical protein electrophoresis
unit and protein transfer apparatus 2.8 - - 2.8
Minor Equipment
4 Minor equipment like rocking
shaker, Minor equipment like
rocking shaker,freezer racks (x3),
flow cell for cytometer etc.
2.2 - - 2.2
5 Total 33 - - 33
Justification for the proposed equipment: Real time PCR machine: This project involves
extensive analysis of alterations in gene expression by quantitative real time PCR. Therefore,
a dedicated real time PCR machine is required for this extensive analysis. Bioruptor
Standard sonication device is required for the sonication of many DNA samples at time
during Chromatin Immuno precipitation (ChIP) experiments for accurate and reproducible
results.
Gel electrophoresis apparatus are required in this project as it extensively involves SDS-
PAGE and western blotting; these are also required routinely in the lab. Rocking shaker is
required for western blot. For FACS analysis capillary is required as our current capillary is
broken and cannot be used for the FACS experiment proposed in the current project. All the
proposed small equipments are bare minimum requirement to carry out this project.
B. Recurring
B.1 Manpower
(~in Rs. Lakhs)
Designation Number of
persons
Monthly
Emoluments
1st
Year
2nd
Year
3rd
Year Total
JRF 2 12000 + 30%
HRA 3.75 3.75 3.75 11.25
Total 15,600 3.75 3.75 3.75 11.25
Justification for the manpower requirement: The work proposed involves extensive
western blotting, site-directed mutagenesis, Chromatin immune precipitation, real time qPCR
19 | P a g e
and RT-qPCR, Si RNA mediated silencing, over-expression of histone mutants. In addition,
immunological assays such as ELISA, oxidative burst as well as FACS. Some of these assays
should be established and must be carried out within the three year time period. Therefore, at
least two JRF are required fulltime for three years to accomplish all the proposed goals.
B.2 Consumables
~ Rs. in Lakhs
S. No. Item Year 1 Year 2 Year 3 Total
1 Kits such as first cDNA
strand syntheis kit, SYBR
green kit, ChIP assay kit,
Monocyte isolation kit, site
directed mutagenesis kit etc.
4.5 2.5 1.4 8.4
2 Cell culture consumables and
plastic ware such as cell
lines, media, FBS,
lipofectamine,
oligofectamine,
nucleofection kits,siRNA,
cell culture dishes, etc.
3 3.5 1.5 8
3 Enzymes and PCR
consumables such DNA
polymerase, LR clonase, BP
clonase, dNTPs etc.
3 2 0.6 5.6
4 Fluorescent dyes - - 0.5 0.5
5 Cytokine ELISA kits such as
TNF-α, IL-12, IL-10 etc.
- - 1.5 1.5
6 Antibodies for histone
modification, iNOS, AKT,
ERK etc. and Western
blotting consumables such as
ECL, X-ray films etc.
4.3 2.5 1.5 8.3
7 ChIP-Seq 5 - - 5
8 Biochemicals 2.2 2 0.6 4.8
9 Miscellaneous like LPS,
IFNγ, oligonucleotides etc.
2 1.5 0.4 3.9
Total 24 14 8 46
B.2 Justification for costly consumables: Fine bio-chemicals, molecular biology reagents,
antibodies, Real-Time assay kits, ELISA kits, site directed mutagenesis kits are expensive
and will be imported. ChIP-Seq analysis is very expensive but is very crucial for the successf
of this project. So, the proposed budget is bare minimum to fulfill the objectives outlined in
this study.
20 | P a g e
B.3 TRAVEL
(For all Investigators and will be divided in equal portions)
N BUDGET (~ in Rs. Lakhs)
N 1st Year 2nd Year 3rd Year Total
Travel 0.25 0.25 0.25 0.75
Justification for intensive travel: The budget for travel will be used to attend relevant
conferences, present the results of the work at national symposia by the laboratory personnel,
co-investigator and the principal investigators.
B.4 CONTINGENCIES
(For all Investigators and will be divided in equal portions)
N (~in Rs. Lakhs)
N 1st Year 2nd Year 3rd
year Total
Other costs/Contingency
costs 1.00 1.00 1.00 3.00
Justification for specific costs under other costs: The proposed budget for other
costs/contingency will be used towards charges for DNA sequencing to verify clones,
synthesizing oligonucleotides. It will also be used for buying books, photocopying,
communication, writing manuscripts, bearing publication costs and other miscellaneous work
of the two investigators. Therefore, it is a minimum of only Rs 50,000 per investigator.
BUDGET ESTIMATES: SUMMARY
~ Rs. in Lakhs
Item Year 1 Year 2 Year 3 Total
A. Non-recurring
(Equipment and accessories)
33 - - 33
B.1 Manpower 3.75 3.75 3.75 11.25
B.2 Consumables 24.00 14.00 8.00 46.00
B.3 Travel 0.25 0.25 0.25 0.75
B.4 Contingency 1.00 1.00 1.00 3.00
B.5 Overhead 1.00 1.00 1.00 3.00
Grand Total (A + B) 63.00 20.00 14.00 97.00
Justification of overheads: This amount is required provide laboratory space, infrastructure,
IT facilities, library facilities etc.
21 | P a g e
PART V: EXISTING FACILITIES
Resources and additional information
1. Laboratory: Laboratory space has been provided at the Institute of Life Sciences
Building at Gachibowli, Hyderabad.
a) Manpower: No manpower exists for this project
List of facilities being extended by parent institution(s) for the project implementation.
A. Infrastructural Facilities:
Sr.
No.
Infrastructural Facility Yes/No/ Not required
Full or sharing basis
1. Workshop Facility Yes
2. Water & Electricity Yes
3. Laboratory Space/ Furniture Yes
4. Power Generator Yes
5. AC Room or AC Yes
6. Telecommunication including e-mail & fax Yes
7. Transportation No
8. Administrative/ Secretarial support Yes
9. Information facilities like Internet/ Library Yes
10. Computational facilities Yes
11. Animal/ Glass House Yes
12. Any other special facility being provided None
B. Equipment available with the Institute/ Group/ Department/ Other Institutes for the
project:
Equipment
available with
Generic Name of Equipment Model, Make & year
of purchase
Remarks
PI's Department 1. Cell culture facility
2. Cold Room
3. Micro Centrifuges
4. Table top centrifuges
5. High Speed Microfuge
6. Sonicator
7. Victor 3 Multi-label plate reader
8. Liquid Nitrogen Storage Tank
9. Flow Cytometer
10. Spectrophotometer
11. Gel documentation system
12 Luminometer
13. Orbital shaking Incubators
14. -86 Freezers
15. UV Cross linker
16. Nucleofector
CRP
Blue star
Eppendorf, Kubota
Eppendorf, Kubota
Thermo Sorvall RC6
Plus
Sonics
Perkin Elmer
Thermo Fisher Sc.
Guava, USA
Perkin Elmer
Wilver
Berthold
New Brunswick
Thermo
GE Health Care
Amaxa/Lonza
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
In use
22 | P a g e
PART VI: DECLARATION/CERTIFICATION
It is certified that
a) the research work proposed in the scheme/project does not in any way duplicate the work
already done or being carried out elsewhere on the subject.
b) the same project proposal has not been submitted to any other agency for financial
support.
c) the emoluments for the manpower proposed are those admissible to persons of
corresponding status employed in the institute/university or as per the Ministry of Science
& Technology guidelines (Annexure-III)
d) necessary provision for the scheme/project will be made in the Institute/University/State
budget in anticipation of the sanction of the scheme/project.
e) if the project involves the utilization of genetically engineered organisms, we agree to
submit an application through our Institutional Biosafety Committee. We also declare that
while conducting experiments, the Biosafety Guidelines of the Department of
Biotechnology would be followed in toto.
f) if the project involves field trials/experiments/exchange of specimens, etc. we will ensure
that ethical clearances would be taken from concerned ethical Committees/Competent
authorities and the same would be conveyed to the Department of Biotechnology before
implementing the project.
g) it is agreed that any research outcome or intellectual property right(s) on the invention(s)
arising out of the project shall be taken in accordance with the instructions issued with the
approval of the Ministry of Finance, Department of Expenditure, as contained in
Annexure-V.
h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same is
signed and enclosed.
i) the institute/university agrees that the equipment, other basic facilities and such other
administrative facilities as per terms and conditions of the grant will be extended to
investigator(s) throughout the duration of the project.
j) the Institute assumes to undertake the financial and other management responsibilities of
the project.
Signature of Executive Authority
Signature of Principal Investigator: Date:
Date:
Signature of Co-Investigator
Date:
23 | P a g e
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Name: Dr Devyani Haldar.
Designation: Principal Research Scientist
Department/Institute/University: Institute of Life Sciences
Date of Birth: 7th
January, 1971 Sex (M/F): F
Email: [email protected]; [email protected]
Phone: +91 (40) 66571500
Fax: +91 (40) 66571581
Education (Post-Graduation onwards & Professional Career)
Sl
No. Institution
Place
Degree
Awarded
Year Field of Study
1. Jawaharlal Nehru
University, New
Delhi, India
Master of
Science in
Biotechnology
1992-1994 Biotechnology
2. Indian Institute of
Science, Bangalore,
India
Ph.D. in
Biochemistry
1994-2001 Biochemistry
3. LGRD, NICHD,
National Institutes of
Health (NIH),
Bethesda, MD
20892, USA
Postdoctoral
Visiting Fellow
2002- 2006 Role of yeast Sirtuin
Hst4 in DNA
damage response.
Position and Honors
Position and Employment (Starting with the most recent employment)
Sl No. Institution
Place
Position From
(Date)
To (date)
Institute of Life Sciences,
University of Hyderabad
Campus, Hyderabad, India.
Senior Research
Scientist
Nov. 2006 April
2011
Institute of Life Sciences,
University of Hyderabad
Campus, Hyderabad, India.
Principal Research
Scientist
May 2011 To date
24 | P a g e
Honors/Awards
Post-doctoral visiting fellowship from National Institutes of Health (NIH), USA (2002-
2006).
Department of Atomic Energy (DEAE), India, Fellowship (1999-2001)
Council for Scientific and Industrial Research (CSIR) - Senior Research
Fellowship (1996 – 1999)
Council for Scientific and Industrial Research (CSIR), India - Junior Research Fellowship
(1994 – 1996)
Department of Biotechnology (DBT), India, Fellowship (1992 – 1994)
Professional Experience and Training relevant to the Project
Post-doctoral visiting fellowship from National Institutes of Health (NIH), USA (Jan
2002- Oct 2006): Worked with yeast Sirtuin Hst4 (its role in DNA damage response)
B. Publications (Numbers only) ..........7.......
Books : .........None........... Research Papers, Reports : 7..General articles : None
Patents : ........None.................Others (Please specify) : None
Selected peer-reviewed publications (Ten best publications in chronological order)
1. Rahul Kumar Vempati and Devyani Haldar (2012) DNA damage in the presence of chemical
genotoxic agents induce acetylation of H3K56 and H4K16 but not H3K9 in mammalian cells. Mol
Biol Rep 39 (2), 2055
2. Mohosin Layek, Syam Kumar Y., Aminul Islam, Ravikumar Karavarapu, Amrita Sengupta,
Devyani Haldar,
K. Mukkanti, Manojit Pal
(2011) Alkynylation of N-(3-iodopyridin-2-
yl)sulfonamide under Pd/C–Cu catalysis: a direct one pot synthesis of 7-azaindoles and their
pharmacological evaluation as potential inhibitors of sirtuins. Med. Chem. Comm. 2, 478.
3. Rahul Kumar Vempati, Ranveer Jayani, Notani D., Amrita Sengupta, Galande S and Devyani
Haldar (2010) p300 mediated acetylation of histone H3 lysine 56 functions in DNA damage response
in mammals J. Biol. Chem. 285 (37), 28553-28564.
4. Devyani Haldar and R.T. Kamakaka (2008) Schizosaccharomyces pombe Hst4 functions in DNA
damage response by regulating histone H3 K56 acetylation. Eukaryotic Cell 7(5), 800-813.
5. Devyani Haldar and R.T. Kamakaka (2006) tRNA genes as chromatin
barriers Nat Struct Mol Biol 13 (3), 192-193.
6. *C. Adams, *Devyani. Haldar and R.T. Kamakaka (2005) Construction and characterization of
a series of vectors for S. pombe Yeast 22 (16), 1307-1314. (*Equal contribution by both authors)
25 | P a g e
7. Devyani Haldar, Samir Acharya and Rao, M.R.S. (2002) A novel structure-specific endonuclease
activity associated with polypyrimidine-tract binding (PTB) related protein from rat testis.
Biochemistry 41, 11628-11641.
List maximum of five recent publications relevant to the proposed area of work.
1. Rahul Kumar Vempati and Devyani Haldar (2012) DNA damage in the presence of chemical
genotoxic agents induce acetylation of H3K56 and H4K16 but not H3K9 in mammalian cells. Mol
Biol Rep 39 (2), 2055
2. Rahul Kumar Vempati, Ranveer Jayani, Notani D., Amrita Sengupta, Galande S and
Devyani Haldar (2010) p300 mediated acetylation of histone H3 lysine 56 functions in DNA
damage response in mammals J. Biol. Chem. 285 (37), 28553-28564.
3. Devyani. Haldar and R.T. Kamakaka (2008) Fission yeast Sirtuin Hst4 functions in DNA
damage response by regulating histone H3 K56 acetylation. Eukaryot Cell 7(5):800-813.
C. Research Support
Ongoing Research Projects:
Sl
No.
Title of Project Funding Agency Amount Date of sanction
and Duration
1.
A supA Suppressor screen to uncover
novel functions of fission yeast
S. pombe Sirtuin Hst4
Department of
Biotechnology
(RGYI), Govt. of
India
22.3
lakhs
March 2010-
2013
3 years
2 A yeast based screen for discovery
of novel Sirtuin inhibitors as anti-
cancer agents. DBT grant no.
Department of
Biotechnology,
Govt. of India
32.52 March 2011-
2013
2 years
Completed Research Projects (State only major projects of last 3 years) None
Place : Signature of Investigator
Date :
26 | P a g e
Co-Investigator:
Name: Dr. Kishore Parsa
Designation: Senior Research Scientist
Institute: Institute of Life Sciences, Hyderabad
Date of Birth: 31-Aug-1975. Sex (M/F) Male SC/ST: N/A
Email: [email protected]; [email protected]
Phone: +91 (40) 66571500
Fax: +91 (40) 66571581
Education (Post-Graduation onwards & Professional Career)
S.
No.
Institution
Place
Degree
Awarded
Year Field of Study
1 Acharya NG Ranga Agricultural
University, Hyderabad
M.V.Sc. 2001 Animal Genetics and
Breeding
2 Texas A&M University-Kingsville,
USA
M.S. 2004 Biochemistry
3 The Ohio State University, USA Ph.D. 2007 Biochemistry
Position and Honors
Position and Employment (Starting with the most recent employment)
S. No. Institution
Place
Position From To
1 Institute of Life Sciences,
Hyderabad
Senior Scientist 2009 To date
2 Matrix Laboratories Ltd,
Hyderabad
Assistant Manager 2008 2009
Honors/Awards
2001 Prize for second best poster at Texas Academy of Sciences, USA
2001-04 Welch scholarship at Texas A&M University-Kingsville, USA
2001-04 Research Assistantship at Texas A&M University-Kingsville, USA
2003-04 Teaching Assistantship at Texas A&M University-Kingsville, USA
2004-05 Program fellowship at The Ohio State University, USA
2005-07 Research Assistantship at The Ohio State University, USA
2007 Best poster award, IGP first annual symposium, USA
2011 Department of Atomic Energy Young Scientist Award (Basic Sciences),
Mumbai, India
27 | P a g e
Professional Experience and Training relevant to the Project
Dr. Kishore Parsa is currently a senior research scientist leading a team of biologists at the
Institute of Life Sciences and has an overall 5 years of research experience post PhD. During
his MS (at Texas A&M University-Kingsville) and PhD (at The Ohio State University) he
was trained in cell biology, molecular biology, immunology and biochemistry and gained the
required skill set. During his PhD, he focused on dissecting signaling pathways playing a
critical role in host pathogen interactions; particularly he worked to understand macrophage
activation by a gram-negative pathogen Francisella novicida. During his entire research
career, he authored 28 articles (and 2 patents) including 21 relevant to the current research
project demonstrating the expertise essential to successfully execute the proposed project.
B. Publications
Research Papers: 28 Patents: 2
Research articles relevant to the proposed project
1. Rajaram MV, Butchar JP, Parsa KV, Cremer JT et al. Akt and SHIP modulate
Francisella escape from the phagosome and induction of the Fas-mediated death
pathway. PLoS One. 2009 Nov 20;4(11):e7919.
2. Parsa KV, Butchar JP, Rajaram MV, Gunn JS, Schlesinger, LS and Tridandapani, S.
Francisella gains a survival advantage within mononuclear phagocytes by
suppressing host IFN response. Mol. Immunol. 2008 Jul; 45(12):3428-37.
3. Parsa KV, Butchar JP, Rajaram MV, Cremer JT and Tridandapani, S. The Tyrosine
kinase Syk promotes the phagocytosis of Francisella through the activation of Erk.
Mol. Immunol. 2008 May;45(10):3012-21.
4. Butchar JP, Parsa KV, Marsh CB and Tridandapani S. IFN gamma enhances IL-23
production during Francisella infection of human monocytes. FEBS Lett. 2008 Apr
2;582(7):1044-8.
5. Henning, LN, Azad, AK, Parsa KV, Crowther, JE, Tridandapani, S, and Schlesinger,
LS. Pulmonary surfactant protein A regulates TLR expression and activity in human
macrophages. J Immunol. 2008 Jun 15;180(12):7847-58.
6. Butchar JP, Rajaram MV, Ganesan LP, Parsa KV, Clay CD, Schlesinger, LS,
Tridandapani, S. Francisella tularensis induces IL-23 production in human
monocytes. J. Immunol. 2007;178:4445-4454.
7. Parsa KV, Ganesan LP, Rajaram MV et al. Macrophage pro-inflammatory response
to Francisella novicida infection is regulated by SHIP. PLoS Pathog. 2006;2:e71
8. Butchar JP, Parsa KV, Marsh CB and Tridandapani S. (2006) Negative regulators of
Toll-like receptor 4-mediated macrophage inflammatory response. Current
Pharmaceutical Design. 2006;12(32):4143-53.
9. Rajaram MV, Ganesan LP, Parsa KV et al. Akt/Protein kinase B modulates
macrophage inflammatory response to Francisella infection and confers a survival
advantage in mice. J Immunol. 2006;177:6317-6324.
28 | P a g e
10. Henning, LN, Azad, AK, Parsa KV, Crowther, JE, Tridandapani, S, and Schlesinger,
LS. Pulmonary surfactant protein A regulates TLR expression and activity in human
macrophages. J Immunol. 2008 Jun 15;180(12):7847-58.
Additional significant contribution to the field
1. Adepu R, Rambabu D, Prasad B, Meda CL, Kandale A, Rama Krishna G, Malla
Reddy C, Chennuru LN, Parsa KV*, Pal M*. Novel thieno[2,3-d]pyrimidines: their
design, synthesis, crystal structure analysis and pharmacological evaluation. Org
Biomol Chem. 2012, Aug 7;10(29):5554-69. *Equal senior authorship
2. Kumar MP, Kumar KS, Meda CL, G. Reddy GR, D. Rambabu, K. Shiva Kumar, R.
Kapavarapu, K. Krishna Priya, Chennubhotla KS, Banote RK, Kulkarni P, Parsa
KV* and Pal M*. (Pd/C-mediated) coupling-iodocyclization-coupling strategy in
discovery of novel PDE4 inhibitors: A new synthesis of pyzolopyrimidines. Med
Chem Comm In Press. *Equal senior authorship
3. Shyamsunder Reddy T, Shiva Kumar K, Meda CL, Kandale A, Rambabu D, Rama
Krishna G, Hariprasad C, Venugopala Rao V, Venkataiah S, Malla Reddy C, Naidu
A, Dubey PK, Parsa KV, Pal M. Conformationally restricted novel pyrazole
derivatives: Synthesis of 1,8-disubstituted 5,5-dimethyl-4,5-dihydro-1H-
benzo[g]indazoles as a new class of PDE4 inhibitors. Bioorg Med Chem Lett. 2012
Mar 14. [Epub ahead of print]
4. Siva Kumar K, Mahesh Kumar P, Sreenivasa Rao V, Jafar AA, Meda CL,
Kapavarapu R, Parsa KV, Pal M. A new cascade reaction: concurrent construction of
six and five membered rings leading to novel fused quinazolinones. Org Biomol
Chem. 2012 Apr 21;10(15):3098-103. Epub 2012 Mar 9.
5. Kumar KS, Kiran Kumar S, Yogi Sreenivas B, Gorja DR, Kapavarapu R, Rambabu
D, Rama Krishna G, Reddy CM, Basaveswara Rao MV, Parsa KV, Pal M. C-C bond
formation at C-2 of a quinoline ring: Synthesis of 2-(1H-indol-3-yl)quinoline-3-
carbonitrile derivatives as a new class of PDE4 inhibitors. Bioorg Med Chem. 2012
Apr 1;20(7):2199-207. Epub 2012 Feb 16.
6. Gorja DR, Shiva Kumar K, Kandale A, Meda CL, Parsa KV, Mukkanti K, Pal M.
Design and synthesis of 4-alkynyl pyrazoles as inhibitors of PDE4: A practical access
via Pd/C-Cu catalysis. Bioorg Med Chem Lett. 2012 Apr 1;22(7):2480-7. Epub 2012
Feb 13.
7. Ram Reddy T, Rajeshwar Reddy G, Srinivasula Reddy L, Jammula S, Lingappa Y,
Kapavarapu R, Meda CL, Parsa KV, Pal M. Montmorillonite K-10 mediated green
synthesis of cyano pyridines: Their evaluation as potential inhibitors of PDE4. Eur J
Med Chem. 2012 Feb;48:265-74. Epub 2011 Dec 21.
8. Kumar PM, Kumar KS, Mohakhud PK, Mukkanti K, Kapavarapu R, Parsa KV, Pal
M. Construction of a six-membered fused N-heterocyclic ring via a new 3-component
reaction: synthesis of (pyrazolo)pyrimidines/pyridines. Chem Commun (Camb). 2011
Nov 10. [Epub ahead of print]
9. Parsa KV, Pal M. Preclinical development of dipeptidyl peptidase IV inhibitor
alogliptin: a brief overview. Expert Opin on Drug Discov, 2011 , (15): 855-869
10. Pal S, Durgadas S, Nallapati SB, Mukkanti K, Kapavarapu R, Meda CL, Parsa KV,
Pal M. Novel 1-alkynyl substituted 1,2-dihydroquinoline derivatives from nimesulide
29 | P a g e
(and their 2-oxo analogues): A new strategy to identify inhibitors of PDE4B. Bioorg
Med Chem Lett. 2011 Aug 19. [epub ahead of print]
11. Reddy GR, Reddy TR, Joseph SC, Reddy KS, Reddy LS, Kumar PM, Krishna GR,
Reddy CM, Rambabu D, Kapavarapu R, Lakshmi C, Meda T, Priya KK, Parsa KV,
Pal M. Pd-mediated new synthesis of pyrroles: their evaluation as potential inhibitors
of phosphodiesterase 4. Chem Commun (Camb). 2011 Jul 21;47(27):7779-81.
12. Kumar KS, Kumar PM, Kumar KA, Sreenivasulu M, Jafar AA, Rambabu D, Krishna
GR, Reddy CM, Kapavarapu R, Shivakumar K, Priya KK, Parsa KV, Pal M. A new
three-component reaction: green synthesis of novel isoindolo[2,1-a]quinazoline
derivatives as potent inhibitors of TNF-α. Chem Commun (Camb). 2011 May
7;47(17):5010-2.
13. Kodimuthali A, Gupta R, Parsa KV, Padala LP, Pal M. Evaluation of Novel 7-
(hetero)aryl-substituted Pyrazolo[1, 5-a]pyrimidines as Phosphodiesterase-4
Inhibitors. Lett Drug Des Discov . (7): 402-408.
14. Malladi S, Parsa KV¥, Bhupathi D, Rodríguez-González MA, Conde JA, Anumula P,
Romo HE, Claunch CJ, Ballestero RP, González-García M. Deletion mutational
analysis of BMRP, a pro-apoptotic protein that binds to Bcl-2. Mol Cell Biochem.
2011 May;351(1-2):217-32. ¥Equal first authorship
Research Support
Ongoing Research Projects:
SR/FT/LS-131/2009 (DST) Parsa (PI) 02/07/12-01/07/15
Molecular analysis of the functional role of miR-7 in the β-cells
Role: PI
BT/PR14123/Med/29/193/2010 (DBT) Parsa (PI) 10/07/10-10/06/13
Understanding the role of PHLPP in IFNγ-mediated innate immune responses
The goal of this project is to decipher the molecular basis of PHLPP, a Ser/Thr phosphatase,
in modulating the innate immune responses of macrophages induced by IFNγ.
Role: PI
2010/20/37B/5/BRNS/2564 (DAE) Parsa (PI) 03/02/11-03/01/14
Identification of novel Akt-interacting partners and investigation of their role in LPS-induced
macrophage inflammatory response
The objective of this project is to understand “Akt-proximal” signaling and study its role in
modulating macrophage inflammatory responses.
Role: PI
30 | P a g e
37(1438)/10/EMR-II (CSIR) Parsa (PI) 11/01/10-10/31/13
Molecular analysis of the role of PHLPP1 in LPS-induced macrophage inflammatory
response
The aim of this study is to examine the function role of PHLPP in regulating endotoxin
induced macrophage inflammatory response.
Role: PI
Place : ILS, Hyderabad Signature of Investigator
Date :
