| Research
Summary
Our
research interests are focused in four areas: defining the roles of
the the PU.1 transcription factor partners critical for normal and
abnormal differentiation of myeloid cells, elucidating the role of
newly cloned cyclin D-binding myb-like protein (DMP1) transcription
factor isoforms in differentiation of normal and malignant hematopoietic
cells, understanding HIV-1 protease inhibitor resistance through molecular
evolution, and the use of HIV-1 vectors to deliver novel viral inhibitors
to human hematopoietic cells to impart an anti-HIV-1 effect.
   
Transcription
Factors: PU.1 and DMP1
PU.1
Defining
PU.1 domain functions
PU.1,
an ets transcription factor family member, is only expressed in hematopoietic
(blood) cells. PU.1 gene-disrupted mice are devoid of B and dendritic
cells, monocytes/ macrophages, and mature neutrophils, but not T cells.
Therefore, PU.1 is necessary for dictating monocyte/ macrophage and
dendritic cell commitment and differentiation, and for neutrophil
differentiation. PU.1 not only has a role in development, but we and
others have shown that it is also required for regulating genes necessary
for monocyte/ macrophage and neutrophil growth and function. Finally,
there is evidence that partial disruption of PU.1 may have a role
in the development of specific leukemias.
PU.1
is composed of three domains, an N-terminal transactivation domain,
a PEST domain, and a C-terminal DNA binding domain. To define which
parts of PU.1 promote myeloid development and function, we have utilized
PU.1 gene-disrupted hematopoietic cells that are obtained from our
PU.1 null mouse, followed by HIV-1 vector delivery of PU.1 wild type
or domain-mutants to restore PU.1 function and allow assessment of
development and function. This approach allows identification of the
PU.1 domains required for monocyte and granulocyte differentiation
and function. This strategy allows a proteomics based methodology
to isolate and characterize transcription factors that interact with
PU.1 at various stages of development and function.
DMP1
The
role of newly cloned cyclin D-binding myb-like protein (DMP1) transcription
factor isoforms in differentiation of normal and malignant hematopoietic
cells
Hematopoietic
(blood) cancer often originates from inactivation and deregulation
of the control of gene expression. The cyclin D-interacting myb-like
protein (DMP1) transcription factor regulates positively human p14ARF
(also referred to as ARF or p16ARF in humans and p19ARF in mouse)
and CD13/ Aminopeptidase N (APN) expression, thus playing a role in
cell-cycle control, differentiation and function of hematopoietic
and non-hematopoietic cells. ARF is critical for the positive regulation
of p53 (and thus a tumor suppressor), which in turns controls cellular
proliferation and modulates apoptosis. We have recently identified
two novel and developmentally expressed human DMP1 splice variants
(termed b and g), of which one of these proteins (b) functions as
a dominant-negative regulator of the originally reported DMP1 protein
(now termed a). We are currently investigating the biological roles
of the various isoforms in normal myeloid and leukemic development.
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Proteomics
Functional
Proteomics of PU.1
Proteomics,
the global investigation of the protein population expressed by a
genome, cells, or tissue types, makes use of biochemical and physical
methods. Proteomics is complementary to studies of gene expression
and changes by analysis of cellular mRNA abundance. Analysis of mRNA
abundance provides useful information about the cell state and the
activity of genes. Protein-based cellular analysis quantifies the
final product, rather than intermediate messengers, of the control
of cell development and function.
As
mentioned, PU.1 is required for myeloid commitment and development.
PU.1 is also critical for regulation of cytokine receptors required
for survival and expansion of blood cells. Over expression of PU.1
causes red blood cell cancers, and conditional expression of PU.1
in adult mice leads to an early differentiation block. One of the
types of leukemias, termed acute myeloid leukemia, is blocked in myeloid
development, which could be attributed to differential expression
of and/or mutations in PU.1. To investigate this question, we are
identifying the protein network involved in hematopoietic development
in normal and abnormal (no, some, increased, or mutant) PU.1 expression
conditions. Analysis of protein modifications involved in absence
and presence of PU.1 and the interacting partners of PU.1 would enable
us to understand the role of PU.1 in the development of specific blood
cell types, and certain cancers.
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HIV-1 Vector Delivery Systems
Gene-delivery
to control HIV-1 infection in hematopoietic cells
We
have generated improved HIV-1 vectors that are capable of sustained
gene expression of chemokine receptor intrabody, siRNA, and ribozyme
genes in many primary human cell types. These HIV delivery systems
contain a reporter gene and elements to enhance gene expression and
prevent silencing. Currently, we are evaluating the biological and
virological effects of CCR5-intrabody genes and other novel cellular
and HIV knockdown genes in hematopoietic stem and CD4 T cells.
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HIV-1 PROTEASE
RESISTANCE
Understanding
the Molecular Evolution of HIV-1 Protease Resistance
Viral
protease is responsible for processing the viral proteins that are
implicated in producing infectious virus. Blocking protease function
inhibits viral production and reduces viral spread. Protease inhibitors
suppress HIV-1 replication to mostly undetectable levels in patients.
However, HIV-1 variants evolve that escape drug-treatment by developing
resistance.
To
better understand the sequence of protease structural changes required
for the development of inhibitor resistance, we have generated a panel
of protease inhibitor resistant proteases varying in their degree
of resistance. These protease mutants are used to structurally and
biochemically define determinates that impart resistance. To define
structural changes in protease during the development of resistance,
RNA aptamer libraries are being produced to probe structure and peptide
substrate libraries will be used to define the biochemistry of substrate
specificity.
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Transcription Factors -
Proteomics - Lentiviral Delivery
Vectors - HIV-1 Protease |
