Regulation of Plasmodium falciparum transcription

Project: Research project

Description

Project Summary.
The long-term goals of the proposed work are to understand the molecular mechanisms
involved in transcriptional regulation in the malaria-causing parasite Plasmodium falciparum.
During the malaria infection, the red blood cell stage of Plasmodium development is responsible
for all of the clinical manifestations of this disease. This developmental stage is characterized by
a cyclical 48-hour growth and replication phase that must be exquisitely regulated at the
transcriptional level to maximize developmental efficiency and fidelity. The mechanisms used
by the parasite to achieve this high level of transcriptional control remain unclear, although
there is mounting evidence that transcription is strictly regulated in this organism. Our
hypothesis is that a recently identified set of putative transcriptional regulators -
the Apicomplexan AP2 (ApiAP2) proteins - are a major family of key
transcriptional regulators during parasite development. The role of ApiAP2 proteins
as transcriptional regulators will be analyzed using biochemistry, molecular biology and
functional genomics tools. The first approach will determine the DNA recognition sequences for
ApiAP2 proteins in vitro using two complementary methods. One method is a PCR-based
selective enrichment of ligands by exponential enrichment (SELEX) using recombinant ApiAP2
proteins and a random library of putative DNA binding sites. Alternatively, a protein binding
microarray (PBM) will be used to hybridize ApiAP2 proteins to a comprehensive collection of
double stranded oligonucleotides. The second approach will be to establish the in vivo role of
ApiAP2 proteins by 1) modulating transcriptional levels of these putative regulators and 2)
generating knockout parasite lines. The effects of these genetic modifications will be assayed
globally using DNA microarrays to detect changes in gene expression. Next, the binding of
ApiAP2 proteins to DNA will be measured in vivo by chromatin immunoprecipitation with DNA
microarray detection (ChIP-chip). These results will define the gene sets directly regulated by
the ApiAP2 proteins and will be compared to the in vitro binding results in Aim 2 as well as to
prior gene expression data to establish a model for stage-specific transcriptional regulation
during parasite development. Finally, we plan to establish and experimentally verify a peptidebased
signaling motif that will allow us to predict the active nuclear localization of Plasmodial
proteins. The ApiAP2 proteins are the first large family of putative transcription factor domains
identified in the genome of P. falciparum and hold great promise for antimalarial intervention
since there are no mammalian counterparts to these proteins.
StatusFinished
Effective start/end date7/15/081/31/15

Funding

  • National Institutes of Health: $162,449.00
  • National Institutes of Health: $391,025.00
  • National Institutes of Health: $390,606.00
  • National Institutes of Health: $185,501.00
  • National Institutes of Health: $394,250.00
  • National Institutes of Health: $233,159.00

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Plasmodium falciparum
Parasites
Proteins
Malaria
Oligonucleotide Array Sequence Analysis
Plasmodium
Antimalarials
Gene Expression
Protein Array Analysis
Chromatin Immunoprecipitation
Genomics
Gene Library
Protein Binding
Oligonucleotides
Biochemistry
Molecular Biology
Transcription Factors
Erythrocytes
Binding Sites
Gene Expression Regulation