CIITA promoter I and isoform I expression and function in cells of the myeloid lineage Open Access

Zinzow-Kramer, Wendy Michelle (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/3f462625p?locale=en
Published

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

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files