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Immunoglobulin loci span relatively vast regions of chromatin. The human IgH locus spans 1250 kilobases, IgK locus spans 1820 kilobases, and the IgL spans 1050 kilobases. During V-DJ or V-J rearrangement, the distant V regions have to be localized in the vicinity of the D or J regions. This is most likely achieved by looping of the DNA between these regions. We use Fluorescence In Situ Hybridization to visualize these regions for a looping conformation. We are investigating the role of E-proteins in inducing this chromatin conformation.
The following movie files show the difference between a looping and a non-looping conformation as captured by FISH (also shown are lamins present on the inner side of the nuclear envelope.)
Avi format:
Loop
No loop
Quicktime movies:
Loop
No loop
Transcriptional
Control of B cell DevelopmentIt is now understood that the
development of mature B
lymphocytes from multipotent progenitors requires the coordinated
activities of a number of transcriptional regulatory proteins. Among
these are EBF, Pax-5 and E2A. E47 and E12 are basic-helix-loop-helix
(bHLH) transcription factors encoded by the E2A gene that bind DNA
either as homodimers or as heterodimers with other bHLH proteins.
E2A-encoded proteins are involved in the differentiation of a number of
cell types, and are particularly important for lymphocyte development.
In B lineage cells, the bHLH DNA binding activity is composed of E47
homodimers whereas in developing thymocytes, mainly heterodimers of E47
and a related bHLH protein, HEB are present. B-cell development in E2A
and E47-deficient mice is completely arrested prior to immunoglobulin
gene rearrangement. Recent studies have indicated that E2A proteins
function in early B lineage development to regulate B lineage-specific
gene expression as well as B cell survival. Under study is how E2A
proteins are regulated by external stimuli and signaling pathways as
well as how they control lymphocyte survival and aging of developing B
cells.
Regulation of
Immnuglobulin Gene Rearrangement and
Isotype SwitchingE2A proteins also regulate
site-specific DNA recombination. We
have developed a system which demonstrates that both E12 and E47 have
the remarkable ability to promote immunoglobulin VJ recombination in a
human embryonic kidney cell line. Since the extreme N-terminal domain
of E2A has been shown to recruit the co-activator protein complex,
SAGA, which contains histone acetylase activity, it is conceivable that
the E2A proteins regulate recombination by promoting locus
accessibility. How E2A proteins regulate recombination is currently
under study. Possible mechanisms include localized accessibility,
looping, and direct recruitment of RAG proteins. Other systems under
study are the regulation of isotype switching and regulation of
E-proteins during B cell activation.
Regulation
of Helix-Loop-Helix Proteins In Thymocyte
DevelopmentE2A protein expression in
thymocytes is required to initiate
T-cell differentiation. During thymoctye development the E-proteins and
their antagonists, Id2 and Id3, regulate T-lineage specific gene
expression and TCR rearrangement. We have used a combination of the
genetic and biochemical strategies to demonstrate that E2A and Id
proteins act to block thymocyte maturation in the absence of pre-TCR
expression, and that pre-TCR signaling acts to promote development in
part by inhibiting E2A activity. Other systems under study include the
regulation of E-protein activity by TCR signaling, the regulation of
allelic exclusion, the role of E-proteins and Id3 in positive and
negative selection as well as thymocyte survival and aging.
Cancer: Pre-B and
T-Acute Lymphoblastoid Leukemia There is now considerable data
indicating that a number of
pro-B, pre-B and T-ALLs are in large part induced by the mutation
and/or repression of E2A activity.
A. In pre-B ALL, the N-terminal domain of E2A gene is fused to either the C-terminal portion of HLF, a leucine zipper encoding protein, or Pbx, a homeodomain gene product. This particular group of leukemia have suboptimal responses to antimetabolite chemotherapy that is effective for most B-lineage leukemias. Thus it is important to establish novel strategies for the treatment of these particular diseases. As a first approach to this question we have identified a number of down-stream targets for E2A/Pbx-1. One of these is a novel growth factor, named Wnt-16, which in turn through an autocrine loop activates the Wint signaling pathway in pre-B cells. We suggest that the constitutive activation of Wnt-16 in pre-B cells is a key step which ultimately leads to the development of the leukemia. Work is in progress to determine whether antagonists of Wint, for example secreted forms of Wint receptors, named FrzBs, have the ability to inhibit the activity of Wint-16 and block cell growth. Additionally, we have recently initiated a study to identify small molecules that would specfically inhibit E2A/Pbx1 DNA binding. Once we have obtained positive results they may lead to immediate novel avenues for the treatment of this particular form of pediatric leukemia.
B. Recent work in our laboratory has indicated that the inactivation of E2A is a key step towards the development of T-ALL. Under study are genetic strategies using mice with mutations that block T-cell development at defined stages into an E2A-deficient background and hence would suggest explanations for the mechanisms underlying tumor suppression. The screening for regulatory targets of E2A will be a focus of further study. In addition, studies are in progress to test E2A proteins, or perhaps proteins positively regulated by E2A, could be used therapeutically against T-ALL when administered as TAT fusion proteins.
A. In pre-B ALL, the N-terminal domain of E2A gene is fused to either the C-terminal portion of HLF, a leucine zipper encoding protein, or Pbx, a homeodomain gene product. This particular group of leukemia have suboptimal responses to antimetabolite chemotherapy that is effective for most B-lineage leukemias. Thus it is important to establish novel strategies for the treatment of these particular diseases. As a first approach to this question we have identified a number of down-stream targets for E2A/Pbx-1. One of these is a novel growth factor, named Wnt-16, which in turn through an autocrine loop activates the Wint signaling pathway in pre-B cells. We suggest that the constitutive activation of Wnt-16 in pre-B cells is a key step which ultimately leads to the development of the leukemia. Work is in progress to determine whether antagonists of Wint, for example secreted forms of Wint receptors, named FrzBs, have the ability to inhibit the activity of Wint-16 and block cell growth. Additionally, we have recently initiated a study to identify small molecules that would specfically inhibit E2A/Pbx1 DNA binding. Once we have obtained positive results they may lead to immediate novel avenues for the treatment of this particular form of pediatric leukemia.
B. Recent work in our laboratory has indicated that the inactivation of E2A is a key step towards the development of T-ALL. Under study are genetic strategies using mice with mutations that block T-cell development at defined stages into an E2A-deficient background and hence would suggest explanations for the mechanisms underlying tumor suppression. The screening for regulatory targets of E2A will be a focus of further study. In addition, studies are in progress to test E2A proteins, or perhaps proteins positively regulated by E2A, could be used therapeutically against T-ALL when administered as TAT fusion proteins.
