CIITA promoter I and isoform I expression and function in cells of the myeloid lineage Público
Zinzow-Kramer, Wendy Michelle (2012)
Abstract
Abstract
CIITA promoter I and isoform I expression and function in cells of
the myeloid lineage
The major histocompatibility class II transactivator (CIITA) is
controlled by three promoters (pI, pIII, and pIV). Each promoter
encodes a unique first exon, spliced into a common second exon,
resulting in three isoforms that differ only at the N-terminus. The
first exon of isoform I, expressed primarily by myeloid cells
(dendritic cells and macrophages), encodes a region with homology
to a caspase recruitment domain (CARD). The CARD domain is a
protein-protein interaction domain, and is commonly associated with
proteins involved in inflammation and apoptosis. The activity and
function of isoform I has not been well studied, and it has been
suggested that the CARD domain enhances CIITA activity. To address
whether CIITA isoform I has a unique function, a mouse model in
which isoform I was replaced with isoform III was generated. In
addition, a mouse line in which the promoter and first exon of
Ciita pI were deleted was also created. No defects in the
formation of CD4 T cells or in responses to viral or bacterial
challenge were seen in mice lacking isoform I. In addition, in the
pI knockout model, only a slight decrease in Ciita and
MHC-II expression was observed in dendritic cells, and no change in
expression was observed in macrophages. These results suggest that
control of pI is mediated by an unknown distal enhancer element
that is also capable of promoting transcription from pIII. Recent
work in our lab and others has identified a regulatory region, HSS1
(DNase hypersensitivity site 1), which is required for
transcription of pIII in B cells and pIV in IFN-γ induced HeLa
cells. Analysis of chromatin structure at the CIITA locus suggests
that HSS1 may also interact with CIITA pI. In B cells, CIITA pI
silencing is likely due to heavy methylation at this promoter. In
non-hematopoietic cells, pIV is inactive unless IFN-γ induced
transcription factors are present. Thus, HSS1 may be a master
enhancer for CIITA, the activity of which is mediated by
methylation and availability of transcription factors.
Table of Contents
Table of Contents
Abstract
Acknowledgements
List of Figures
Chapter 1: General Introduction
1
I. Adaptive immunity: Major Histocompatibility Class
II
1
II. Regulation of MHC-II expression: The WXY
box
9
III. Regulation of MHC-II expression: CIITA
12
IV. CIITA and expression of non-MHC-II genes
17
V. CIITA gene regulation
20
VI. Protein structure and function of CIITA
23
VII. The CARD domain of CIITA
29
VIII. Aims of the study
33
Chapter 2: Materials and Methods
36
Chapter 3: CIITA promoter I CARD-deficient mice express
functional MHC class II
genes in myeloid and lymphoid compartments
50
Abstract
51
Introduction
52
Results
55
Discussion
79
Acknowledgements
83
Chapter 4: Regulation of CIITA promoter choice
88
Introduction
89
Results
93
Discussion
96
Chapter 5: Discussion
104
I. The CARD domain of CIITA is not required for proper
immunological responses
105
II. CIITA promoter I is not required for MHC-II
expression in myeloid cells
107
III. Concluding remarks
111
Literature Cited
115
Figure List
Chapter 1
Figure 1: MHC-II locus
4
Figure 2: CIITA promoter region
14
Figure3: CIITA protein interactions
26
Figure3: NLR family members
32
Chapter 2
Figure 1: CIITA targeting construct
46
Table 1: Primer sequences used in this chapter
47
Table 2: Speed congenic markers
49
Chapter 3
Figure 1: CIITA targeting construct
57
Figure 2: MHC-II surface expression on KI and KO antigen
presenting cells is similar
to WT
59
Figure 3: Ciita isoform I is not required for expression of
CIITA or MHC-II in cells of the
myeloid lineage
64
Figure 4: 5'RACE maps Ciita expression to promoters III and IV
in KI and KO spDC
66
Figure 5: CIITA isoform I is not required for T cell development
in the thymus
68
Figure 6: CIITA pI KO T cells proliferate normally I response to
antigen
70
Figure 7: Ciita pI KO mice are not more susceptible to
experimentally induced autoimmune
disease
72
Figure 8: CIITA isoform I is not required for resistance to
intracellular bacteria Listeria
monocytogenes
74
Figure 9: The adaptive immune response to an acute LCMV
infection in KO mice is similar
to WT
77
Supplemental Figure 1: CIITA regulated genes do not require
CIITA isoform I
for expression
85
Supplemental Figure 2: 5'RACE sequence results from splenic
dendritic cells
86
Supplemental Figure 3: CIITA mice form antibody secreting B
cells in response to
immunization with influenza
87
Chapter 4
Figure 1: Promoter proximal regions of CIITA
100
Figure 2: Methylation across the CIITA locus
101
Figure 3: Methylation at CIITA pI and pIII
102
Figure 4: 5-azacytidine treatment of A20 and J774
cells
103
Chapter 5
Figure 1: Looping at the CIITA locus
113
Figure 2: Histone modification and transcription factor binding
at the CIITA locus
114
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