Nuclear Transport And Its Effect On Breast Cancer Tumor Cells
Curriculum Vitae.
1. Curriculum Vitae
Name DEVANAND KUMAR
Contact C/o Dr. Swati Saha
Department of Microbiology,
University of Delhi South Campus
Benito Juarez Road, New Delhi-21
Email: d21kumar@yahoo.co.in , krishna21a@gmail.com
Mobile: (+91) 9871833701, (+91) 9968050916
Work and Education
July 2015 to date
May 2013 to June 2015
Research Scientist, Premas Biotech. Pvt. Ltd.
Plot No. 77,, Sector 4, IMT Manesar, Gurgaon, Haryana 122050
Dr. Swati Saha, Associate Professor
DNA replication and Chromatin Biology Laboratory
Department of Microbiology,University of Delhi South Campus
Benito Juarez Road, New Delhi-110021
Research Associate (post-doctoral)
Functional characterization of histone acetyltransferase HAT3 in
Leishmania donovani
Sep 2008 to April 2013 Dr. Swati Saha, Associate Professor
DNA replication and Chromatin Biology Laboratory
Department of Microbiology, University of Delhi South Campus
Benito Juarez Road, New Delhi-110021
Doctor of Philosophy (Ph.D.), Microbiology
Specialization: Molecular and Cellular Biology
Thesis: Identification and characterization of histone acetylases in the
protozoan Leishmania donovani
April 2006 to Aug 2008 Dr. Vinay Kumar Nandicoori
Signal Transduction Laboratory-1
National Institute of Immunology, Aruna Asaf Ali Marg
New Delhi-110067
Junior Research Fellow (JRF)
Specialization: Molecular and Cellular Biology
Aug 2003-Sept 2005 Department of Biotechnology
University of Burdwan, Burdwan, West Bengal
Masters in Science (M.Sc) Biotechnology
Thesis: Mechanistic studies on wild Type and mutant human
Guanylate Binding Protein-1(s)
2. Current Job & Skills Devanand Kumar
Standard Molecular and Cellular Biology techniques
Isolation of genomic DNA, RNA and plasmid
PCR, Cloning of genes, Site Directed Mutagenesis
Maintenance of COS-1, J774A.1 cell lines and Leishmania donovani
Transfection of COS-1 and Leishmania cells
Creation of genomic knockouts in Leishmania
Growth and survival curves
Cell synchronization
FACS
Protein and Antibody-related techniques
Recombinant protein expression and purification
Raising of antibodies in mice and rabbits by subcutaneous injections into animals
ELISA
Peptide competition assays
Isolation of chromatin
SDS-PAGE, Western blotting, Immunoprecipitations
2D gel electrophoresis
FPLC
HPLC
Assays
In vitro kinase assays
Peptide mapping and phosphoramino acid analysis
Metabolic labeling
Electrophoretic Mobility Shift Assay (EMSA)
Acetyl transferase assays
Luciferase assays
Use of software for analysis
Swiss-PDB, Rasmol, Pymol, Chimera
Gene runner, Molecular toolkit, Chromas lite
BLAST, CLUSTAL W, EMBOSS, JalView
Image J
MS Office Word, Excel, PowerPoint, Sigma-Plot etc.
Adobe Illustrator, Adobe Photoshop
3. Ph.D. work: Devanand Kumar
Title: Identification and characterization of histone acetylases in the protozoan Leishmania
donovani
Major findings
Leishmania histone acetyltransferases HATs 1 and 3 are nuclear throughout the cell cycle.
HAT4 is cytosolic throughout the cell cycle but also nuclear in G2/M phase.
HAT3 and HAT4 acetylate the N-terminal tail of histone H4 in vitro.
HAT3 specifically targets the 4th
lysine residue of H4 in vitro.
HAT4 specifically targets the 14th
lysine residue of H4 in vitro.
HAT3 is main mediator of H4K4 acetylation in vivo.
HAT3 is not essential for growth and survival but HAT3 depletion decreases cell viability and
prolongs S phase and G2/M phases of the cell cycle.
HAT3 interacts stably with DNA polymerase processivity factor PCNA in cell extracts.
Summary
Post-translational modifications on histones regulate various cellular processes like DNA
replication, recombination, transcription and repair. Two types of histone modifications have been
found to regulate DNA replication – acetylation and methylation. Histone acetylation helps
hro ati to a hieve a ore ope stru ture a et lati g the a i o groups of lysine side-chains,
which aids the recruitment of incoming replication factors thus helping the DNA to replicate. The
acetylation of histones is catalyzed by histone acetyltransferases which transfer an acetyl group from
acetyl-CoA to lysine residues of histones. Histone acetylation statuses are regulated by histone
acetyltransferases (HATs) and histone deacetylases (HDACs). While the HATs are associated with the
activation of chromatin, the HDACs are associated with chromatin repression as deacetylation of
these lysine residues strengthens histone-DNA interactions.
Of the two types of HATs (Type A and Type B), acetylations mediated by Type B HATs,
occurring prior to chromatin assembly, play a crucial role in histone deposition on chromatin;
acetylations mediated by Type A HATs mostly occur on histones after they have been assembled into
chromatin, and regulate various aspects of DNA metabolism. Type A HATs broadly fall into three
families – the GNAT, MYST and CBP/p300 families. While the sequences of eukaryotic histones and
the modifications they carry are largely conserved from yeast to mammals, while trypanosome
histones are highly divergent in sequence. The identification of histone modifications in
Trypanosoma brucei and Trypanosoma cruzi has revealed that the N-terminal tails of H2A, H3 and H4
carry several acetylation (and methylation) marks. Although specific histone modification marks
have not been previously identified in Leishmania species, as the sequence of Leishmania histones
are highly conserved with those of Trypanosoma histones, it may be inferred that the PTMs are likely
to be conserved.
Leishmania genome encodes four histone acetyltransferases of the MYST family, named as
HATs 1-4. HATs 1-3 are reported to be present across all trypanosomatids, while HAT4 is absent in
T.brucei. As part of my Ph.D. work I cloned and partially characterized three of these HATs: HAT1,
HAT3 and HAT4. Genes for HAT1, HAT3 and HAT4 were cloned by amplification using Leishmania
genomic DNA as template, and the authenticity of the genes were confirmed by DNA sequencing.
HAT1 and HAT4 were successfully expressed in E.coli in soluble form but all our efforts to express
HAT3 in soluble form in E.coli failed. Antibodies were raised against the purified recombinant HAT1
and HAT4, and these antibodies were used to assess expression of the native proteins in Leishmania
whole cell extracts. To determine the subcellular localization of the proteins at different stages of
the cell cycle all three HATs were expressed in fusion with GFP in Leishmania promastigotes. We
found that while HAT1 and HAT3 localize to the nucleus throughout the cell cycle, HAT4 localizes to
4. Ph.D. work: Devanand Kumar
the cytosol at all stages and is also found in the nucleus in post-mitotic cells. A fluorescence based
HAT assay was used to determine substrate specificities of these HATs. Histone H4 was found to be
the substrate of HAT3 and HAT4, with the target sites of HAT3 and HAT4 being identified as K4 and
K14 respectively. To investigate role of H4K4 acetylation in the cell two tools were generated:
H4acetylK4 modification-specific antibodies and a HAT3 knockout line. Modification-specific
antibodies were raised in rabbit and the antibody specificity was verified by peptide competition
assays. H4K4 acetylation was analyzed using those H4acetylK4 specific antibodies. More or less
equivalent levels of H4K4 acetylation were observed at different stages of Leishmania (log vs
stationary and non-infective procyclics vs infective metacyclics). To examine H4K4 acetylation at
different stages of the Leishmania cell cycle, cells were synchronized using hydroxyurea to arrest
them at the G1/S boundary and then released into S phase. Cell cycle progression was analyzed by
flow cytometry and whole cell lysates were also prepared at the same time points. H4K4 acetylation
was found to occur more or less concomitantly with histone synthesis in S phase.
To examine the role of the H4K4 acetylation event a genomic knockout of HAT3 was created.
The two genomic alleles were replaced stepwise by homologous recombination using suitable drug
resistance markers. The fidelity of the knockouts was verified by PCR amplification across the
deletion junctions using appropriate primers. RT-PCR analyses of RNA isolated from HAT3-null lines
confirmed that HAT3 was no longer expressed in the nulls. Thus, it is evident that HAT3 is not
essential to cell survival. Western blot analyses of lysates isolated from HAT3- nulls revealed the
drastic downregulation of H4K4 acetylation, though residual acetylation was observed. This result
indicates that HAT3 is the main mediator of H4K4 acetylation in vivo. HAT3-null lines showed
decreased cell viability, slower S phase, and prolonged G2/M. The H4K4 acetylation and growth
defects were partially rescued by episomal expression of HAT3 in the HAT3-null lines. H4K4
acetylation in trypanosomes has been believed to be analogous to H4K5 acetylation in higher
eukaryotes. The H4K5 acetylation event in higher eukaryotes has been found to positively modulate
DNA replication. To investigate the possible role of H4K4 acetylation in DNA replication in
Leishmania, we first examined if HAT3 exists as a part of replication factories that are known to exist
across eukaryotes for which PCNA serves as marker. We found HAT3 to co-immunoprecipitate with
PCNA in Leishmania extracts, suggesting that HAT3 may be a part of the replication factories found
in the cell. This aspect was further investigated as part of my post-doctoral work.
Post-doctoral work:
Title: Functional characterization of histone acetyltransferase HAT3 in Leishmania donovani
Major findings
H4K4 acetylation is important for histone deposition into nucleosomes in Leishmania
HAT3 ediates the ell s response to UV-induced DNA damage.
HAT3 ediates its effe t ediati g PCNA s role i DNA repair.
HAT3 mediates PCNA degradation via ubiquitin-proteasome pathway post-UV exposure.
HAT3 mediates PCNA acetylation post-UV exposure.
HAT3 mediated PCNA acetylation marks PCNA for subsequent monoubiquitination, a step
that is essential for Translesion DNA synthesis- based DNA repair pathway.
Summary
Histone post-translational modifications (PTMs) impact various cellular processes, such as
chromatin environment, gene expression, DNA replication and DNA repair. Histone acetylation
events in eukaryotes have been demonstrated to play an important role in regulating transcription,
origin activation and DNA repair, in addition to modulating histone deposition and nucleosome
formation. While histone PTMs and their functional relevance are largely conserved across
eukaryotes from yeast to mammals, trypanosome histones (and consequently their PTMs) are
divergent from acetylation seen in higher eukaryotes. Acetylation of histone H4 at K5 in other
5. Post-doctoral work: Devanand Kumar
eukaryotes has been shown to modulate histone deposition, DNA damage repair, transcriptional
activation and cell cycle progression.
As a part of my Ph.D. work we found that deletion of the histone acetyltransferase HAT3
from Leishmania cells caused the drastic downregulation of H4K4 acetylation. HAT3-nulls displayed
decreased cell viability, slower S phase progression and prolonged G2/M. H4 K5/K12 diacetylation in
Saccharomyces cerevisiae has been shown to be essential for histone deposition into nucleosomes.
To address the question of whether H4K4 acetylation in Leishmania modulates histone deposition,
we isolated the soluble and chromatin-bound protein fractions in wild type Leishmania as well as
HAT3-KO cells. Western blot analyses of soluble and DNA-associated protein fractions showed that
while H4 was predominantly found in the DNA-associated protein fraction in cells expressing HAT3,
it was equally distributed in the fractions of soluble and DNA-associated proteins in HAT3-nulls.
These data suggest that H4K4 acetylation in Leishmania plays an important role in histone
deposition into nucleosomes. Defects in histone deposition in HAT3-nulls may lead to genomic
instability and thus decreased cell viability.
As part of my Ph.D. work we also found HAT3 to exist in a stable complex with the DNA
polymerase clamp protein PCNA in cell extracts, suggesting a possible role for HAT3 in regulating
DNA replication. We now examined the possibility of HAT3 helping load PCNA onto chromatin. Our
analyses revealed that HAT3 most likely helps load PCNA onto chromatin in normally growing cells.
To examine the possible effects of deficits in PCNA loading onto chromatin in HAT3-KO cells, wild
type and HAT3-null cells were synchronized using hydroxyurea treatment, released into S phase, and
analyzed for DNA replication at different time-points after release, by pulsing cells with EdU and
examining cells for EdU uptake microscopically. No clear-cut difference in DNA replication pattern
was evident.
DNA polymerase clamp protein PCNA plays a role in mediating DNA replication as well as
DNA repair events. To address the possibility of HAT3 modulating DNA repair, wild type and HAT3-
null cells were exposed to UV radiation and the effect on H4K4 acetylation was examined in
irradiated cells. Western blot analyses of whole cell lysates isolated from irradiated cells revealed a
gradual increase in H4K4 acetylation post-UV irradiation. Levels of unmodified H4 also increased,
signifying synthesis of new histones with concomitant H4K4 acetylation. To directly investigate if
HAT3 plays a role in mediating cell survival after induction of DNA damage, HAT3-KO cells were
subjected to UV irradiation and their growth monitored for 5 days post-UV exposure. We found that
HAT3-KO cells showed poor recovery after UV irradiation compared to wild type cells. In order to
e a i e the role of HAT3 i regulati g the ell s respo se to UV-induced DNA damage, the effect of
UV irradiation on PCNA distribution between soluble and DNA-associated fractions was analyzed in
wild type vs HAT3-null cells, and surprisingly, we found that while PCNA cycled off/on in wild type
cells following UV exposure, it was continuously retained on chromatin in HAT3-nulls. To check if
PCNA cycling pattern in wild type cells was due to its degradation via the ubiquitin-proteasome
pathway, cells were incubated with proteasome inhibitor (MG132) prior to UV exposure, and soluble
and DNA-associated fractions analyzed for PCNA. We observed that while the PCNA distribution
pattern remained unaffected by treatment with proteasome inhibitor in HAT3-nulls, the protein was
markedly retained on chromatin in wild type cells treated with MG132 in comparison with untreated
cells. This indicated that PCNA was being degraded via the ubiquitin-proteasome pathway in wild
type cells but not HAT3-KO cells following exposure to UV. Additionally, a band corresponding to the
monoubiquitinated form of PCNA was also detected in the chromatin-bound fraction of wild type
cells that had been treated with proteasome inhibitor, and importantly, this band was not detected
in HAT3-null cells. These results raised the interesting possibility of the HAT3-PCNA interaction
having a role additional to recruitment of PCNA to chromatin. To examine this possibility we
examined the interaction of HAT3 and PCNA after UV treatment. We found that UV exposure
heightened the interaction of the two proteins.
These findings raised the question of what could be the outcome of a heightened interaction
between HAT3 and PCNA (post-UV). PCNA acetylation has been reported to be acetylated in human
6. Post-doctoral work: Devanand Kumar
cells post-UV exposure, and the HATs responsible for this acetylation were recently identified as the
human CBP/p300. The possible role of HAT3 in PCNA acetylation after UV exposure was explored. It
was found that while basal levels of acetylated PCNA were evident in almost all cases,
hyperacetylated PCNA was prominent only in immunoprecipitates from UV-irradiated wild type cells
that had been incubated with proteasome inhibitor before irradiation. This acetylated form of PCNA
migrated at the same position as monoubiquitinated PCNA, suggesting the presence of an acetylated
form of monoubiquitinated PCNA in wild type cells (but not HAT3-KO cells) in which the ubiquitin-
proteasome pathway had been inhibited. The results obtained suggest that PCNA is strongly
acetylated in response to UV, and HAT3 plays a major role in mediating the acetylation of PCNA in
response to UV-induced DNA damage. This is the first report linking a HAT-mediated acetylation of
PCNA to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the
mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes.
List of Scientific Publications
1. Kumar D and Saha S. (2015) HAT3 mediated acetylation of PCNA precedes PCNA
monoubiquitination following exposure to UV in Leishmania donovani. Nucleic Acids Res.
(doi.10.1093/nar/gkv431).
2. Parikh A*, Kumar D*, Chawla Y*, Kurthkoti K, Khan S, Varshney U, Nandicoori VK. (2013)
Development of a new generation of vectors for gene expression, gene replacement, and
protein-protein interaction studies in mycobacteria. Appl Environ Microbiol. 79 (5):1718-29.
*contributed equally to this work
3. Kumar D*, Kumar D*, Saha S. (2012) A highly basic sequence at the N-terminal region is essential
for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani.
Microbiology 158(Pt7):1775-82.
*contributed equally to this work
4. Kumar D, Rajanala K, Minocha N, Saha S. (2012) Histone H4 lysine 14 acetylation in Leishmania
donovani is mediated by the MYST-family protein HAT4. Microbiology 158 (Pt 2):328-37.
5. Minocha N, Kumar D, Rajanala K, Saha S. (2011) Characterization of Leishmania donovani
MCM4: expression patterns and interaction with PCNA. PLoS One 6(7):e23107.
6. Minocha N, Kumar D, Rajanala K, Saha S. (2011). Kinetoplast morphology and segregation
pattern as a marker for cell cycle progression in Leishmania donovani. J Eukaryot Microbiol.
58(3):249-53.
7. Khan S, Nagarajan SN, Parikh A, Samantaray S, Singh A, Kumar D, Roy RP, Bhatt A, Nandicoori VK.
(2010 ) Phosphorylation of enoyl-acyl carrier protein reductase InhA impacts mycobacterial
growth and survival. J Biol Chem. 285 (48):37860-71.
8. Kumar D, Minocha N, Rajanala K, Saha S. (2009) The distribution pattern of proliferating cell
nuclear antigen in the nuclei of Leishmania donovani. Microbiology 155(Pt 11):3748-57.
9. Pawan Kumar*, Kumar D *, Parikh A, Ranaware D, Gupta M, Singh Y and Nandicoori VK. (2009)
The Mycobacterium tuberculosis protein kinase K modulates activation of transcription from the
promoter of mycobacterial monooxygenase operon through phosphorylation of the
transcriptional regulator VirS. J Biol Chem. 284(17):11090-9.
*contributed equally to this work
7. List of Scientific Publications Devanand Kumar
10. Vomastek T, Iwanicki MP, Burack WR, Tiwari D, Kumar D, Parsons JT,Weber MJ & Nandicoori
VK. (2008) ERK2 phosphorylation sites and docking domain on the Nuclear Pore Complex
protein Tpr cooperatively regulate ERK2-Tpr interaction. Mol Cell Biol. 28(22):6954-66.
References
Dr. Swati Saha
Associate Professor
Dept. of Microbiology
University of Delhi South Campus
Benito Juarez Road
New Delhi-21
Email -ss5gp@yahoo.co.in
Prof. J.S. Virdi
Dept. of Microbiology
University of Delhi South Campus
Benito Juarez Road
New Delhi-21
virdi_dusc@rediffmail.com
Dr. Vinay Kumar Nandicoori
Scientist
National Institute of Immunology
Aruna Asaf Ali Marg
New Delhi.-67
Email - vinaykn@nii.res.in
Dr. Apurba Kumar Sau
Scientist
National Institute of Immunology
Aruna Asaf Ali Marg
New Delhi.-67
Email - apurba@nii.res.in