2805241829
IDENTIFICATION OF REGULATORY ELEMENTS IN
THE CDS GENE LOCUS
Amd Martin Hostert
Research Thesis submitted for the degree of doctor of philosophy
of the University of London
Division of Molecular Immunology
National Institute for Medical Research
Mill Hill
London
ProQuest Number: 10014291
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A
b s t r a c t
The m urine CD8 molecule is a disulphide linked heterodim er expressed on thymocytes and some T cells. It is composed of a CD8a and a CD8(3 chain, both members of the imm unoglobulin gene superfamily, w ith a total m olecu lar mass of 70 kilodalton. Its expression is developm entally regulated and plays an im portant role during thymocyte developm ent and cytotoxic lym phocyte effector function. The m urine CD8 gene locus consists of the CD8a gene downstream of the CD8P gene separated by 36 kilobases with the two genes organized in the same transcriptional orientation.
The CD8 gene locus was cloned from genomic cosmid and PI libraries. A 80kb PI clone, ICRFP703B21317 (Pl-5), was isolated that contains the CD8 genes and flanking DNA sequences.
DNasel hypersensitivity analysis carried out using DNA isolated from DNasel treated thymocyte nuclei detected four clusters of DNasel hypersen sitive sites (DNasel-HSS) in the CD8 gene locus (Cl - CIV). Analogous analy sis using DNA isolated from DNasel treated liver nuclei show ed three of the four clusters to be thymocyte specific (CII - CIV).
Transgenic mice carrying the cloned CD8ap genomic locus and contain ing the identified DNasel-HSS clusters express the transgenic C D 8a in a developm entally regulated, tissue specific and CD8 T cell subset specific manner. N orthern blot analysis of CD8P RNA show ed that the transgenic CD8P gene was also expressed only in those tissues which were positive for the endogenous CD8p RNA.
A
c k n o w l e d g e m e n t s
F irst I w o u ld like to th an k m y su p erv iso r D im itris K ioussis for his invaluable help and guidance throughout this project. W ithout his attention and support this study w ould not have been possible. Further I w ould like to thank Rose Zamoyska for invaluable help w hen I started this project.
I w ould also like to thank Mauro Tolaini w ho not only generated trans genic mice together w ith Dimitris but who also had the m isfortune to have his bench and desk space next to mine and Chris Atkins for his invaluable technical assistance w ith the four-colour FACS stainings.
C
o n t e n t s
Ab s t r a c t m
Ac k n o w l e d g e m e n t s IV
Co n t e n t s V
Lis t o f Fig u r e s XI
Ab b r e v ia t io n s XVI
In t r o d u c t i o n 1
The Im m une System 2
B Cells 4
T Cells 5
Generation of diversity 6
Costimulation 7
T cell development 7
M olecular Biology Of CD4 A nd CDS 12
The Cell Surface Molecule CDS 12
CDSa cDNA structure and genomic organization 13
CD8p cDNA structure and genomic organization 15
The Cell Surface Molecule CD4 22
CD4 A nd CD8 In T Cell D evelopm ent A nd Function 24
Interaction of CD4 and CDS w ith MHC molecules 24
A role for CD4 and CDS in intracellular signalling 27
A role for CD4 and CDS in T cell developm ent 29
CD4 knock out mice 30
CDSa knock out mice 30
CDSp knock out mice 31
R egulation Of Gene Expression 33
Eukaryotic gene structure 33
The prom oter 33
The basal transcriptional machinery 34
Activated transcription 35
Enhancers 35
The CD4 enhancer 36
Mechanisms of enhancer function 36
Silencers 3S
The CD4 silencer 39
C hrom atin Structure A nd Gene R egulation 42
Histories and nucleosome structure 42
Chrom atin and transcription 44
DNasel hypersensitive sites 46
Control O f CDS Gene Expression 48
CDSa gene regulatory elements 48
Analysis of the hCDSa prom oter and DNasel-HSS 50
CDSP gene expression regulatory elements 51
Post-transcriptional regulation of CDSa' and CDSp 53
Project Aims 55
Ma t e r ia l s a n d M e t h o d s 56
G eneral 57
Chemicals and reagents 57
Bacteriological 57
Bacteriological cultures 57
Competent bacteria and cell transform ation 57
Small scale plasm id DNA isolation 58
Large scale plasm id DNA isolation 59
Genomic DNA preparation 60
DNasel sensitivity 61
DNA restriction digests 63
Agarose gel electrophoresis 63
Extraction of DNA from agarose gels 64
DNA fragment preparation and microinjection 64
Blotting and hybridization 65
Southern blots 65
Slot blots 66
DNA probe labelling 67
Filter hybridizations 67
Cosmid and PI library preparation 68
H ybridization of libraries 69
A utoradiography 70
Membrane stripping 70
RNA 70
Extraction of total RNA from tissues 70
N orthern blots 71
Sequencing 73
Exonuclease III deletions 73
Screening of exonuclease III deletions 73
DNA sequencing 74
Flow Cytometry 75
Re s u l t s An d Di s c u s s i o n 77
C loning O f The CDS Gene Locus 78
Isolation and characterization of cosmid and PI clones 79
Colinearity of Pl-5 with the genomic CDSap locus 94
Summary 102
D N asel Sensitivity A nalysis O f The CDS Gene Locus 105
Identification of DNasel hypersensitive site clusters 106
Tissue specificity of DNasel hypersensitive site clusters 122
Finemapping of DNasel hypersensitive site cluster CII 131
Finemapping of DNasel hypersensitive site cluster CIII 138
Summary 141
Analysis O f Pl-5 DNA Sequences In Transgenic Mice 144
Antibody specificity 145
Summary 162
D eletion Analysis Of D N asel H ypersensitive Site C luster CIII 164
cosCDSa and Pl-1 transgenic mice 165
Analysis of CD8-CIII transgenic mice 171
CD8a-CIII-123 transgenic mice 171
CD8a-CIII-12 transgenic mice 179
CD8a-CIII-l transgenic mice 185
CD8a-CIII-2 transgenic mice 193
CD8a-LCR transgenic mice 201
CD2-CIII-123 transgenic mice 209
Appearance of transgene positive T cells coincides 217 w ith positive selection
Summary 220
Co n c l u d i n g Re m a r k s 230
Re f e r e n c e s 235
L
is t
o f
F
ig u r e s
In t r o d u c t io n
Figure I T lymphocyte development 11
Figure II S tru ctu re of the cell surface m olecules C D 8a, 21 CD8a' and CD8p
Results
Figure 1 Organization of the CD8 gene locus 81
F ig u re2 R e s tric tio n a n a ly sis of c o sm id s cos2-5.3 84 (cos p), cosCD8a (cos a) and plasmid Pl-5
Figure 3 Hybridization of CD8a cDNA probe to plasm ids 87 cos2-5.3 (cos p), Pl-5 and cosCD8a (cos a)
Figure 4 Hybridization of CD8P cDNA probe to plasm ids 89 cos2-5.3 (cos p), Pl-5 and cosCD8a (cos a)
Figure 5 H ybridization of l.Zkb XM-Scfll probe to plas- 91 m ids COS2-5.3 (cos P), Pl-5 and cosCD8a (cos a)
Figure 6 H ybridization of 0.5kb HindUl probe to plasm ids 93 cos2-5.3 (cos p), Pl-5 and cosCD8a (cos a)
Figure 7 H ybridization of vector probe to plasm ids cos2- 96 5.3 (cos p), Pl-5 and cosCD8a (cos a)
Figure 9 C o lin earity of clone P l-5 w ith genom ic D N A 101 (0.9kb HindlU probe)
Figure 10 Restriction enzyme m ap of the CD8 gene locus 104
Figure 11 Identification of D N asel hypersensitive sites in 108 cluster I
Figure 12 Restriction m ap of the C D 8 a/p intergenic region 110 sh o w in g p ro b e s u se d in id e n tify in g D N a se l
hypersensitive site clusters
Figure 13 Identification of D N asel hypersensitive sites in 112 cluster II
Figure 14 Identification of D N asel hypersensitive sites in 115 cluster III
Figure 15 Identification of D N asel hypersensitive sites in 117 cluster IV
Figure 16 Identification of additional DNasel hypersensitive 119 sites in cluster IV
Figure 17 Restriction m ap of the CD8 gene locus show ing 121 DNasel hypersensitive site clusters
Figure 18 H ybridization of liver albumin probe to DNA iso- 124 lated from DNasel treated liver nuclei
Figure 20 DN asel hypersensitive site cluster CII is thym o- 128 cyte specific
Figure 21 DNasel hypersensitive site cluster Gin is thymo- 130
cyte specific
Figure 22 DNasel hypersensitive site cluster CIV is thymo- 133 cyte specific
Figure 23 DNasel hypersensitive site cluster CIV is thymo- 135 cyte specific
Figure 24 Finem apping of HSS cluster CII D N asel hyper- 137 sensitive sites
Figure 25 Finem apping of FISS cluster CIII DNasel hyper- 140 sensitive sites
Figure 26 S p e c ific ity of a n ti-C D 8 a /L y t-2 .1 a n d a n ti- 147 CD8a/Lyt-2.2 antibodies
Figure 27 Southern blot analysis of Pl-5.1 and Pl-5.5 trans- 150 genic mice
Figure 28 P l-5 tra n s g e n ic m ice e x p re ss th e tra n s g e n ic
CD8a/Lyt-2.2 allele specifically on CD8 positive 154 cells
Figure 29 Expression of the transgenic CD8a/Lyt-2.2 trans- 156 gene is developmentally regulated
Figure 31 Expression of the transgenic C D 8 a/p genes is tis- 161 sue specific in Pl-5.5 transgenic mice
Figure 32 Location of cosCDSa and plasm id Pl-1 167
Figure 33 DNasel-HSS cluster CIII deletion analysis 170
Figure 34 S o u th e rn b lo t a n aly sis of CD8a-CIII-123 a n d 173 CD8a-Cin-12 transgenic mice
Figure 35 Expression of CD 8a/Lyt-2.2 in the periphery of 175 CD8a-Cin-123 transgenic mice
Figure 36 E xpression of C D 8 a/L y t-2 .2 in the th y m u s of 178 CD8a-CIII-123 transgenic mice
Figure 37 Expression of CD8a/Lyt-2.2 in the periphery of 181 CD8a-Cin-12 transgenic mice
Figure 38 E xpression of C D 8 a/L y t-2 .2 in the th y m u s of 184 CD8a-CIII-12 transgenic mice
Figure 39 Southern blot analysis of CD8a-Cni-l transgenic 187
mice
Figure 40 Expression of CD8a/Lyt-2.2 in the periphery of 189 C D 8a-C in-l transgenic mice
Figure 41 E xpression of C D 8 a /L y t-2 .2 in the th y m u s of 192 C D 8a-C in-l transgenic mice
Figure 43 Expression of CD8a/Lyt-2.2 in the periphery of 197 CD8a-CIII-2 transgenic mice
Figure 44 E xpression of C D 8 a/L y t-2 .2 in the th y m u s of 200 CD8a-CIII-2 transgenic mice
Figure 45 Southern blot analysis of CD8a-LCR transgenic 203 mice
Figure 46 E xpression of C D 8 a/L y t-2 .2 in the th y m u s of 206 CD8a-LCR transgenic mice
Figure 47 Expression of CD 8a/Lyt-2.2 in the periphery of 208 CD8a-LCR transgenic mice
Figure 48 Southern blot analysis of CD2-CIII-123 transgenic 211 mice
Figure 49 Expression of hCD2 in the periphery of CD2-CIII- 213 123 transgenic mice
Figure 50 Expression of hCD2 in the thym us of CD2-CIII- 216 123 transgenic mice
Figure 51 T ransgene ex p ressio n coincides w ith p o sitiv e 219 selection
A
b b r e v ia t io n s
AFP a-fetoprotein
AFC Antigen presenting cell
APS Am m onium persulphate
ATP Adenosinetriphosphate
BCR B cell receptor
bp Basepair
BSA Bovine serum albumin (Fraction V)
CAT Chloroamphenicol transferase
CD2 Cluster of differentiation antigen 2 CD3 Cluster of differentiation antigen 3 CD4 Cluster of differentiation antigen 4 CDS Cluster of differentiation antigen 8
cDNA Complementary DNA
cm Centimetre
cpm Counts per m inute
CRE cyclic AMP response element
CREB CRE binding protein
CTL Cytotoxic T lymphocyte
CTP Cytosinetriphosphate
d d H 2 0 Double distilled water
DMSO Dimethylsulphoxide
DN CD4"CD8" double negative
DNA Deoxyribonucleic acid
DNasel Deoxyribonuclease I
DNasel-HSS DNasel hypersensitive site DP CD4‘*"CD8''" double positive
DTE Dithioerythritol
DTT Dithiothreitol
EDTA Ethylene diamine tetraacetic acid
EGTA Ethyleneglycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid
FACS Fluorescent activated cell sorting
ECS Fetal calf serum
GXP Guanosinetriphosphate
hCD2 H um an CD2
hCDSa H um an CDSa
HY Male specific antigen
lEL Intraepithélial T lymphocytes
Ig Immunoglobulin
IgH Ig heavy chain
IPTG Isopropyl-p-D-thiogalactopyranoside
kb Kilobase
kDa Kilodalton
kHz Kiloherz
KOAc Potassiumacetate
LCR Locus control region
LTR Long terminal repeat
Mb Megabase
MEL Mesenteric lymphnode T lymphocytes
mg Milligram
MHC Major histocompatability complex
ml Millilitre
m m Millimetre
MMTV Mouse m am m ary tum or virus
mRNA Messenger ribonucleic acid
NaCitrate Sodiumcitrate
NaOAc Sodiumacetate
nm Nanometre
NH^OAc Ammoniumacetate
NK N atural killer
nt Nucleotide
O.D. Optical density
Pl-5 ICRFP703B26205
PEL Peripheral blood lymphocytes
PBS Phosphate buffered saline
pc Post coital
PEG Polyethylene glycol
Pg Picogram
PMA Phorbol 12 - myristate 13 - acetate
PMSF Phenylmethylsulphonylflouride
PVP Polyvinylpyrolidine
RNA Ribonucleic acid
RNApol II RNA polymerase II
RNaseA Ribonuclease A
rpm Revolutions per m inute
SI SI nuclease
SAR Scaffold attachment region
SDS Sodium dodecylsulphate
sig Surface Ig
SSC Sodium chloride, sodium citrate
SP CD4'^CD8' or CD4"CD8^ single positive TAB Tris acetate EDTA electrophoresis buffer
TBE Tris borate EDTA electrophoresis buffer
TBP TATA box binding protein
TdT Terminal deoxynucleotidyl transferase
TEMED N,N,N',N'-tetra-methyl-ethylenediamine
IC R T cell receptor
Tris Tris (hydroxymethyl) aminomethane
TTP Thymidinetriphosphate
3'UT 3' untranslated
UTP Uridinetriphosphate
u.v. Utraviolet
V Volts
v / v Volume per volume
w / v Weight per volume
w /w Weight per weight
This thesis examines DNA regulatory elements that control the expres sion of the genes encoding the heterodim eric cell surface m olecule CD8. Since CDS plays an im portant function in cytotoxic T cells of the im m une sys tem, this introduction covers the topics of the im m une system, T cell develop m ent, structure and function of the CDS molecule and the regulation of gene expression.
The Im m une System
It is w idely thought that mammals have evolved an im m une system so as to defend themselves from potentially dangerous organisms such as virus es, bacteria, fungi and other parasites and thus from 'danger to se lf. To fulfil th e se fu n c tio n s th e im m u n e sy ste m h as to be c ap a b le of re c o g n isin g pathogens and foreign molecules called antigens and therefore distinguish 'foreign' from 'se lf. Furthermore, in order to protect the host efficiently it has to be able to m ount a rapid and specific imm une reaction against previously encountered antigens (adaptive imm une response). The above functions are ascribed to two broadly defined mechanisms, innate im m unity (present at all times and nonspecific) and the adaptive specific im m une response which is induced by foreign antigen giving rise to long lasting protection.
The adaptive immune response on the other hand is m ediated mainly by cells which express receptor molecules on their cell surface (see below) capable of recognising foreign antigen and then m ounting a specific imm une response to the inducing molecule. These cells, know n as lymphocytes, are present in almost all tissues of the body and circulate constantly through the blood. A large proportion of these cells is located prim arily in the lym phoid organs which can be distinguished into prim ary and secondary organs. The major prim ary lym phoid organs are the bone m arrow and the thym us, the secondary lym phoid organs are the spleen, lym ph nodes and Peyer's patches. It is in the prim ary lymphoid organs where lymphocyte developm ent takes place w hilst most of the cell interactions leading to im m une responses against antigen occur in the secondary lymphoid organs.
Lymphocytes can be divided into phenotypically distinct subpopula tions that carry out specialized functions. Broadly, the distinction is m ade betw een B and T cells. Whilst both cell types are capable of recognising anti gen via cell surface receptors (see below), they do so in a different manner. B cells recognize whole or "native' antigen, whereas T cells recognize antigen only in the form of short peptides (8-20 amino acids) presented on cell surface m o le c u le s k n o w n as th e M ajor H is to c o m p a ta b ility C o m p le x (M H C) (Zinkemagel and Doherty, 1979; Babbitt et al., 1985; Buus et al., 1986).
include those that are derived from endogenous proteins, b u t also those of intracellular pathogens such as viruses (Brodsky and Guagliardi, 1991). MHC class I is present on the cell surface of almost all cell types so that any cell that m ay be infected w ith a pathogen can display the antigenic peptide to effector cells. Additionally, the ubiquitous expression of MHC class I allows for the presentation of peptides derived from 'abnorm al' endogenous proteins gener ated by m utations or of novel proteins such as tum or antigens in transform ed cells. In contrast, MHC class II, expressed prim arily on cells of the imm une system, presents peptides derived from the proteolytic digestion of internal ized antigens which then complex w ith MHC class II molecules (Davidson et al., 1991; Peters et al., 1991; Tulp et al., 1994).
B cells
captured and processed antigen, these B cells then differentiate into plasma cells which secrete antibodies specific for the recognized antigen.
T cells
T cells can be subdivided into two groups on the basis of their cell sur face m ark er expression. Two such m arkers are CD4 an d CD8. CD4 is expressed on 70% of peripheral m ature T cells which recognize antigenic pep tides in the context of MHC class II and are mainly of a T helper cell pheno type. They are thought to activate macrophages and to stimulate B cells that have bound antigen to differentiate into antibody producing plasm a cells. The rem aining 30% of T cells express the CD8ap heterodimer, are m ainly of a cytotoxic phenotype and recognize antigen in the context of MHC class I (Swain, 1983).
G eneration of diversity
C ostim ulation
Simple recognition of antigen or peptide bound to MHC is usually not sufficient for activation of naive T cells that have previously not encountered antigen. These cells require a second signal from another cell (Bretscher and Cohn, 1970). T cells are thought to receive the second signal from so called professional antigen presenting cells such as B cells, macrophages and den d ritic cells. C ostim ulatory signals are delivered th ro u g h the interaction betw een cell surface molecules on the T cell and the antigen presenting cell. Such costim ulatory molecules include the co-receptors CD4 and CDS (see below). A further costimulatory molecule know n as CD28, present on T cells has been show n to be a major contributing molecule in costimulation interact ing w ith a ligand called B7 (Harding et al., 1992; H arding and Allison, 1993; Lenschow and Bluestone, 1993; Lenschow et al., 1993; Seder et al., 1994).
T cell developm ent
T cell developm ent occurs in the thym us, an organ consisting of two lobes th at is located above the heart. In ad d itio n to o th er bone m arrow derived cells each of the lobes consists of epithelial cells interspersed w ith developing thymocytes. The blood vessels divide each lobe into num erous lobules w ith an outer cortical region called the thymic cortex and an inner m edulla. The tightly packed cortex contains mainly cortical epithelial cells w ith long branched dendritic processes rich in MHC class II expression and a large num ber of im m ature thymocytes that are in close contact w ith these cells (Wekerle et al., 1980; Kyewski and Kaplan, 1982; Kyewski et al., 1982; van Ewijk, 1991). The medulla, less tightly packed w ith thymocytes than the cortex, contains MHC class I and MHC class II positive epithelial cells w ith broader processes than those found in the cortex.
called negative selection (Kappler et al., 1987; Kappler et al., 1988; Smith et al., 1989). One m odel to explain positive and negative selection is the affinity model. This model proposes that thymocytes whose TCR binds M H C /p ep tid e w ith high affinity are elim inated by negative selection, w hereas low affinity interaction results in positive selection (Sprent et al., 1988; Schwartz, 1989). Only a small percentage of the DP thymocytes complete the selection process (Egerton, 1990) and differentiate into MHC class II or MHC class I restricted m ature thym ocytes having dow nregulated the CD8 or CD4 co receptor respectively. These CD4 or CD8 SP cells migrate into the peripheral lym phoid organs and establish the T cell pool (Figure I). The co-receptor phe n o ty p e is correlated w ith the specificity of the TCR for MHC class (von Boehmer et al., 1989). Two m ain models have been proposed that address how TCR and co-receptor specificity are matched. The stochastic m odel sug gests that co-receptor downm odulation is random and that only those cells that have a combination of a TCR capable of recognising an MHC molecule in conjunction w ith the appropriate co-receptor (MHC class II and CD4 or MHC class I and CD8) survive (Chan, 1993; Davis et al., 1993). Those which have a m ism atch betw een TCR and co-receptor recognition for MHC die. In con trast, the instructive model postulates that TCR specificity for MHC deter m ines which of the two co-receptors is dow nregulated (Borgulya et al., 1991; Robey et al., 1991).
FIGURE I: T CELL DEVELOPMENT
E a r l y
O GE N I T
Bone Marrow
M olecular B iology Of CD4 And CDS
As describ ed above, the cell surface m olecules CD4 an d CD8 are expressed on peripheral T cells in a m utually exclusive m anner whilst under going a complex pattern of expression in the thymus. This part of the intro duction focuses on the structure, molecular biology and function of the CD8 and to a lesser extent the CD4 molecules.
The cell surface m olecule CDS
Murine CD8 has been described as two separate antigens, Lyt-2 (CD8a) and Lyt-3 (CD8P) (Cantor and Boyse, 1975) and were found to be encoded by separate genes, CD8a and CD8p, which m ap to chromosome two in hum an and six in the mouse. The CD 8a/Lyt-2 and CD8p/Lyt-3 genes are closely linked to each other as well as to the immunoglobulin (Ig) K chain gene locus (Itakura et al., 1972; Gottlieb, 1974; Gibson et al., 1978; Sukhatme et al., 1985; Gorm an et al., 1988).
Using antibodies directed against the CD8a and CD8p chains two alle les have been defined for each of the CD8a and CD8p chains. The alleles encoding these chains have been designated Lyt-2.1 and Lyt-2.2 (CD8a) and Lyt-3.1 and Lyt-3.2 (CD8P) (Boyse and Old, 1971). The serological differences are due to nucleotide differences betw een the alleles that result in amino acid substitutions affecting the extracellular portions of the CD8a and CD8p mole cules (see below: Serological differences betw een the CD 8a and CD8p alleles).
In contrast to conventional T cells such as those isolated from lym ph nodes or spleen, intraepithélial T lymphocytes (lEL) located in epithelial cell layers exhibit a m arkedly different expression pattern of apTCR and the CD8 co-receptor molecules. Whilst mesenteric lym ph node cells (MEL) and lEL contain approximately equal num bers of CD3"^ cells, the ratio of CD4 to CD8 cells (3:1 in MEL), is almost reversed in lEL w ith 70% expressing CD8 and <10% expressing CD4 (Maloy et al., 1991). In contrast to CD8"'" MEL which alm ost exclusively express the CD8ap heterodimer, only a m inority of lEL express the CD8aP heterodim er and have a norm al Vp repertoire similar to MEL. The rem ain d er carry C D 8 a a h o m o d im ers an d are th o u g h t to be extrathymically derived (Guy-Grand et al., 1991).
C D 8a cDNA structure and genomic organisation
tid e of 21 am ino acids follow ed by a m atu re p ro tein of 214 am ino acids (Littman et al., 1985; Sukhatme et al., 1985). The extracellular portions of hCD 8a consist of a combination of a 96 amino acid large Ig-V like dom ain and a m em brane proxim al region of 65 am ino acids also called the hinge. This is followed by a transm em brane dom ain of 24 am ino acids th at is of hydrophobic amino acid composition and a 29 amino acid cytoplasmic tail containing basic amino acid residues. M urine CD8a cDNA clones were iso lated by cross-hybridisation using hCD8 cDNA clones as probes (Nakauchi et al., 1985; Zamoyska et al., 1985) and sequence analysis show ed the molecules to have a high structural and sequence homology being 56% and 61% identi cal at the amino acid and nucleotide level respectively (Nakauchi et al., 1985; Zamoyska et al., 1985).
Isolation and transfection of the mouse CD8a gene contained w ithin a 5.5kb Hm dlll fragment showed that the two polypeptides CD 8a and C D 8a' described above arise by translation of alternatively spliced C D 8a mRNA transcribed from a single CD8a/Lyt-2 gene (Zamoyska et al., 1985; Tagawa et al., 1986). Determination of the nucleotide sequence and intron - exon struc ture of the mouse CD8a genomic locus showed that the CD8a mRNA is alter natively spliced to include or exclude exon IV (CD8a or C D 8a' respectively) (Liaw et al., 1986). A lternative splicing of hC D 8a m RN A has also been rep o rte d (Littm an and G ettner, 1987); how ever, it is the tran sm em b ran e dom ain that is removed from hCD8a mRNA and no change in reading frame occurs dow nstream of the alternative splice acceptor site. The resulting pro tein can be secreted by transfected cells (Littman, 1987) although it is not know n if this is also the case in hum ans in vivo.
The genom ic organisation of the h u m an C D 8a gene has also been determ ined and is very similar to that of the mouse. Briefly, the gene consists of six exons and five introns covering approxim ately 7kb in the genome. Similar to the m urine CD8a gene, the exons correspond roughly to the func tional dom ains of the hCD8a protein (Nakayama et al., 1989b; N orm ent et al., 1989).
CDSp cDNA structure and genomic organisation
TCR variable regions. Furthermore and distinct from CD8a, a 12 amino acid segm ent that is extremely similar to the im m unoglobulin joining segm ents w as identified, w ith as m any as 10 out of 12 residues identical to the hum an X-light chain joining sequence Ql) (Gorman et al., 1988). As described above, the Ig-V like dom ain is connected to the transm em brane segm ent of the CD8p protein by a dom ain of 28 amino acids, also called the connecting peptide. Using rat cDNA clones as probes (Johnson and Williams, 1986) m urine CD8P cDNA w as isolated and found to be very similar in its structure and dom ain o rg a n iz a tio n to ra t CD8P w ith 78% of a m in o acid re s id u e s id e n tic a l (Nakauchi et al., 1987a; Panaccio et al., 1987; Blanc et al., 1988; Gorm an et al., 1988). M urine CD8p appears to have only one N-linked glycosylation site in contrast to three identified in rat. As is the case for CD 8a mRNA an alterna tive form of splicing of m urine CD8P mRNA occurs. This event gives rise to a 90bp deletion that removes the transmembrane dom ain and its deletion cor relates w ith alternative splicing to exclude the exon encoding this domain. Thus, the resulting protein product is 162 instead of 192 amino acids in size (Gorman et al., 1988) but it is not known whether this protein is secreted.
The genomic organisation of m urine CDSP was determ ined by several groups (Blanc et al., 1988; Gorman et al., 1988; Nakayam a et al., 1989a) and th e gene w as fo u n d to be p resen t at single copy in the m u rin e genom e (Gorman et al., 1988; Nakayama et al., 1989a). Gorm an and co-workers (1988) cloned the CD8p gene by chromosomal walking from CD8a. Using a cloned cDNA encoding CD8p the genomic organisation of the CD8P gene was deter m ined to be in the same transcriptional orientation compared to CD8a and found to be located approximately 36kb upstream of CD8a. N akayam a et al. (1989) carried o u t a d etailed intron - exon analysis by cloning the CD8P genom ic gene from cosm id libraries of BIO.A (C D 8p/Lyt-3.2) an d AKR (CD8p/Lyt-3.1) origins. Southern blot analysis of BaniHl digested genomic DNA isolated from eleven strains of mice identified a restriction enzym e polym orphism of the CD8p gene (Nakayama et al., 1989). In ten out of the eleven different strains analyzed and including C57B1/6 b u t not C B A /C a mice, a CD8p cDNA probe hybridizes to a 15.5kb genomic BaniHl fragm ent in agreem ent w ith data reported previously by Blanc et al. (1988). In contrast, C 3 H /H e N mice w ere found to carry CD8p coding sequences on a 23kb
m om a cell line could be detected by southern blot analysis (Nakauchi et al., 1987a; Gorm an et al., 1988). Rearrangement of the CD8(3 gene is therefore not necessary for its expression.
In contrast to the single copy CD8p/Lyt-3 gene locus in m ouse, hum ans appear to carry a duplicated CD8p gene (Nakayama et al., 1992). CD8pl has been m apped to chrom osom e two, how ever the chrom osom al location of CD8p2 is still to be determined. Structurally, CD8pl consists of nine exons w h ilst CD8p2 w as found to contain seven. As m entioned above, CD8P2 appears to have arisen from a duplication of CD8pl and sequence analysis show ed that in the coding regions are 98.5% identical. Pulse field gel elec trophoresis analysis indicated that the hCD8a is located dow nstream of the CD8pl gene and are arranged in the same transcriptional orientation.
Serological differences betw een CDSa and CDSp alleles
As described above, both the CD8a and CDSP genes have two serologi cally defined alleles (Lyt-2.1, Lyt-2.2 and Lyt-3.1, Lyt-3.2 for CD 8a and CD8p respectively). In the case of CD8a this difference is the result of a G (Lyt-2.1) to A (Lyt-2.2) nucleotide transition that leads to an amino acid substitution of Valine to Methionine respectively at amino acid position 78 in the m ature pro tein. The allelic difference for CD8p is the consequence of a C (Lyt-3.1) to A (Lyt-3.2) transversion resulting in an amino acid substitution from Arginine to Serine at amino acid position 77.
product from that of the endogenous locus using FACS analysis. (CBA/Ca x C57B1/10)F]^ non-transgenic mice were used as controls in these stainings since they carry and express both the CD8a/Lyt-2.1 and CD8a/Lyt-2.2 alleles (One allele inherited from each of the parents).
FIGURE II: STRUCTURE OF THE CD8a. CDSa" AND CD8p CHAINS
A. Structure of the CD8a chain.
B. Structure of the CD8a' chain. The molecule is identical to that show n in A. w ith the exception of a shortened cytoplasmic tail (see text).
C. Structure of the CD8(3 chain.
A
B
102 102
157 157
193 193
195
220
%
CD8a
CD8a'
147
173
292
The cell surface m olecule CD4
is the case for CD8a and CD8(3 a connecting peptide is located betw een the ]'
and transm em brane domains.
CD4 And CDS In T Cell Development And Function
In general, expression of CD4 and CD8 divides m ature T cells into two subpopulations w ith apparently distinct properties. Thus, the expression of CD4 and CD8 on the surface of peripheral T lymphocytes had initially been correlated w ith T cell function and CD4 positive cells were thought to be of helper and CD8 positive cells of a cytotoxic phenotype (Cantor and Boyse, 1977; R einherz et al., 1979b; Reinherz et al., 1979a; R einherz et al., 1980). H ow ever, the identification of CD4 positive cytotoxic an d CD8 p ositive helper cells (Swain and Panfili, 1979; Swain et al., 1981a; Swain et al., 1981b; Biddison et al., 1982; Krensky et al., 1982a; Krensky et al., 1982b) has now resulted in a correlation of CD8 and CD4 co-receptor expression w ith a recog nition capacity for peptides presented by MHC class I and class II respective ly (Swain, 1983). Therefore, most CD8 SP cells are MHC class I restricted and m ost CD4 SP cells are MHC class II restricted (Engleman et al., 1981; Swain, 1981; M euer et al., 1982a; M euer et al., 1982b).
Interaction of CD4 and CDS w ith MHC m olecules
Furtherm ore they showed that the respective artificial target cells formed con jugates w hen they were incubated together. Direct binding of CD4 to MHC class II w as dem onstrated by Doyle and Strom inger (1987) in experim ents show ing that hum an B cells expressing MHC class II bound to monolayers of transfected cells expressing high levels of CD4. This binding could be inhibit ed by both anti-CD4 and anti-MHC class II monoclonal antibodies.
A d h e sio n of CD4 an d CDS to M HC class II a n d I resp ec tiv e ly is th o u g h t to be due to direct contact betw een the co-receptor and the MHC molecule. M utational analysis of MHC class I molecules suggested that CDS and MHC class I interact at an exposed acidic side loop of the a3 dom ain of the MHC class I molecule (Salter et al., 1990). These experiments also point to one CDSa molecule binding to one MHC class I molecule w hich was con firmed by Gao et al. (1997) who examined the crystal structure of hC D Saa hom odim ers bo u n d to MHC class I (HLA-A2). The region of CDSa th at m akes contact w ith MHC class I appears to be at the loops located at the tops and sides of the Ig-like domains of CDSa (Giblin et al., 1994; Gao et al., 1997). In case of CD4 it is a stru c tu re form ed by th e e x tra ce llu lar D1 an d D2 dom ains (Clayton et al., 19S9; Lamarre et al., 19S9) which were found to bind the p2 dom ain of MHC class II (Cammarota et al., 1992; Konig et al., 1992).
al., 1996), extending the observations of Luescher et al. (1995) w ho originally dem onstrated that CD8 increases the time of interaction betw een a TCR and a p ep tid e/M H C complex.
A possible significance of the interaction between the co-receptors and MHC antigens and the associated increase in interaction betw een TCR and pep tid e/M H C complexes came from studies on the inhibition of T lym pho cyte function by antisera and monoclonal antibodies against the co-receptors. Several experiments have showed that cytotoxicity can be inhibited by anti- CD8 antisera (Nakayama et al., 1979; Shinohara and Sachs, 1979) and anti- CD8 m o n o clo n al a n tib o d ie s (E vans et al., 1981; R ein h erz et al., 1981; L andegren et al., 1982). Similarly, proliferation of CD8 positive T cells in m ixed lym phocyte reactions could also be blocked by anti-CD8 antibodies (H ollander et al., 1980; Englem an et al., 1983). CD4 responses w ere also blockable by anti-CD4 m onoclonal antibodies (Dialynas et al., 1983a). In summary, these experiments were suggesting that the co-receptor molecules are required for increasing the avidity of TCR - peptide/M H C antigen inter actio n s (M acD onald et al., 1982b; M arrack et al., 1983; Sw ain, 1983) as described by Garcia et al. (1996). However, a variability in the efficiency of effector function blocking by anti-CD8 or anti-CD4 antibodies betw een differ ent CTL or CD4 clones was observed (MacDonald et al., 1982a; MacDonald et al., 1982b; Dialynas et al., 1983a). Thus, it is thought that T cells expressing TCRs w ith high affinity for peptide/M H C are less dependent on the interac tion of either CD4 or CD8 w ith p ep tid e/M H C than those expressing TCR w ith low affinity for MHC.
CTL that is specific for fluoresceine (FI) in the context of H2-D^ into a CDS negative cytotoxic cell line. The CTL was capable of lysing both Fl-conjugat- ed fibroblasts and lymphoblasts that expressed high and low levels of H2-D^ respectively In contrast, the transfectant only lysed Fl-conjugated fibroblasts that expressed high levels of MHC. Supertransfection of the TCR transfectant w ith C D Sa restored its ability to lyse Fl-conjugated lym p h o b last targets (Dembic et al., 19S7). This indicated that CDSa contributed substantially to the recognition capacity of the TCRap transfectant. In a similar study Gabert et al. (19S7) transfected a MHC class II restricted T cell hybridom a w ith the TCRa and TCRp chains of a MHC class I restricted TCR. FACS analysis indi cated cell surface expression of the transfected TCR but the transfectant was unresponsive to MHC class I. Indeed, the transfectants only show ed MHC class I specificity w hen co-transfected w ith the C D 8a gene (Gabert et al., 1987). The responsiveness, as assessed by 11-2 release could be blocked by the addition of an anti-CD8a monoclonal antibody indicating the importance of CD 8a in the recognition of MHC by the transfected TCR (Gabert et al., 1987).
A role for CD4 and CD8 in intracellular signalling
The interaction of the cytoplasmic tails of the CD4 and CD8 co-receptor molecules w ith p56^^^ suggests a role for CD4 and CD8 in intracellular sig nalling. Indeed, antibody m ediated cross linking of the TCR w ith either CD4 or CD8 leads to intracellular tyrosine phosphorylation (Gilliland et al., 1991), enhanced Ca^'*' mobilization (Ledbetter et al., 1988) and cellular proliferation (Eichmann et al., 1989). These effects are not observed if crosslinking is car ried o u t w ith CD4 or CD8 co -recep to rs th a t lack p56^^^ b in d in g sites (Chalupny et al., 1991; Collins et al., 1992) consistent w ith the view that the co-receptor brings p56^^^ into the proximity of the TCR and the associated CDS polypeptide complex.
Direct evidence for a signalling function of CD8 during T cell activation has come from several experiments which investigate the role of the CD 8a cytoplasmic tail (Zamoyska et al., 1989; Letourneur et al., 1990). In particular, tailless C D 8a', w hich lacks the p56^^^ binding site, was not as efficient at restoring antigen responsiveness of a T cell hybridom a w hen com pared to the full length CD8a molecule (Zamoyska et al., 1989). In support of these obser vations it was also show n that the ability to restore antigen responsiveness correlated w ith the ability of CD8a to interact w ith p56^^^ (Zamoyska et al., 1989). In the case of CD4 an increase in p56^^^ activity and the associated phosphorylation of the CD3Ç subunit can be induced by antibody crosslink ing of the CD4 molecule (Barber et al., 1989; Veillette et al., 1989).
al., 1993). These mice had a seven fold reduction in peripheral CDS T cells and a five fold reduction in the capacity to generate anti-viral CTL. In addi tion these mice also exhibited impaired selection of an MHC class I transgenic TCR repertoire (Fung-Leung et al., 1993). It is therefore likely th at a sig nalling function through CDSa is not only m ediated by its interaction w ith p56^^^ b u t also through additional, as yet unidentified, proteins.
Likewise, the cytoplasmic tail of CD4 appears to be of importance. A tailless version of CD4 can substitute in vivo for wild type CD4, how ever a reduced num ber of CD4 cells is produced in such mice (Killeen et al., 1993).
Both CD4 and CDS are phosphorylated on serine residues during T cell activation by antigen or phorbol esters (Acres et al., 19S6; Acres et al., 19S7; Blue et al., 19S7). A study by Shin et al. (1990) showed that all three serine residues in the cytoplasmic tail of CD4 are phosphorylated in response to phorbol esters. Substitution of all three residues to alanine resulted in the loss of CD4 phosphorylation (Shin et al., 1990) and in significant im pairm ent in its ability to restore the antigen responsiveness of a T cell hy b rid o m a (Glaichenhaus et al., 1991) whilst the ability of p56^^^ to interact w ith the CD4 cytoplasmic tail was not affected. Phosphorylation of the cytoplasmic tail of CD4 and participation in signal transduction is therefore an im portant part of the T cell activation process. In contrast, inhibition of CDSa cytoplasmic ser ine p h o sp h o ry latio n does n o t ap p ear to have a detrim ental effect on the responses of CDS-dependent transfectants to antigen (Williams et al., 1991).
A role for CD4 and CDS in T cell developm ent
CD4 knock out mice
T ran sg e n ic m ice w ith an in a c tiv a te d CD4 g en e h a v e b e e n g e n e ra te d (Rahem tulla et al., 1991). A lthough T helper cell developm ent is severely impaired, some T helper cells w ith a CD4"CD8" phenotype develop.
C D 8a knock out mice
In contrast, disruption of the CD 8a gene has a m uch m ore dram atic effect. Since CD8p cell surface expression depends upon the presence of the CD 8a protein chain (see below: Post-transcriptional regulation of the C D 8a' and CD8p genes) disruption of the CD8a gene leads to a complete abrogation of CD8 cell surface expression.
M utant mice lacking CD8 expression on the cell surface have been gen erated by disrupting the coding region of the CD 8a/Lyt-2 gene (Fung-Leung et al., 1991). Peripheral cells from these mice were unable to m ount a cytotox ic imm une response against alloantigens. In addition, these mice, prim ed in vivo with vaccinia virus, were unable to m ount a specific cytotoxic imm une response against the virus (Hirsch et al., 1968; Blanden, 1974; Koszinowski and Thomssen, 1975). Analysis of proliferative responses against alloantigen w as studied and showed that the CD4'*' T cells in these mice are functional and can respond to MHC class II alloantigens. In addition, these mice were able to switch VSV-specific antibody isotypes from IgM to IgG, a process that is strictly dependent on T cell help (Gupta et al., 1986).
CDSP knock out mice
Regulation O f Gene Expression
A cell expresses only a limited num ber of genes of its total gene pool. The correct timing of transcription and translation of these genes is critical for the determ ination of the cells' phenotype and thus m ust be tightly controlled. It is thought that the selective transcription of genes is, to a great extent, regu lated by the interaction of proteins binding to gene regulatory DNA elements but is also influenced by chromatin structure. This p art of the introduction focuses on gene regulatory elements, the proteins that bind to these, the struc ture of chrom atin and its role in gene expression.
Eukaryotic gene structure
E u k ary o tic genes are a rra n g ed into cod in g an d n o n -co d in g DNA sequences, exons and introns respectively (Berget et al., 1977; Chow et al., 1977; Jeffreys and Flavell, 1977; Tilghman et al., 1978). In addition to the pro tein coding DNA other im portant gene regulatory elements are associated w ith the genetic unit. These include the prom oter at the 5' end of the gene and other control elements such as enhancers a n d /o r silencers located at vari able distance from the transcriptional initation site, that increase or decrease the tissue specific expression of the linked gene.
The prom oter
precise site of transcription initiation since deletions betw een the TATA box an d the n atu ral initiation site give rise to new transcriptional start points (Grosschedl and Birnstiel, 1980b; Grosschedl and Bimstiel, 1980a; Benoist and Chambon, 1981; Mathis and Chambon, 1981; Kovacs and Butterworth, 1986a; Ko vacs and Butterworth, 1986b). Some prom oters however lack a TATA box m otif b u t also initiate transcription properly and in such prom oters a DNA elem ent called the initiator, of loose consensus sequence, overlaps the precise transcription start site (Weis and Reinberg, 1992; Gill, 1994; Javahery et al., 1994). Further to these critical binding sites, other DNA elements are located in the promoter. These elements appear to play an im portant role in prom ot er function (eg. CCAAT motif, SPl binding sites and CACC box) contributing to tissue specificity and enhancement of transcription initiation by influenc ing the assem bly of the basal tran scrip tio n al m achinery (Ptashne, 1988; Mitchell and Tjian, 1989; Dynlacht et al., 1991). However, they appear to dif fer from elements such as enhancers (see below) since their position relative to the transcriptional initiation site is inflexible (McKnight, 1982).
The basal transcriptional machinery
In order for transcription of a gene to occur, the basal transcriptional m achinery has to assemble at the core prom oter elements. Binding of the transcriptional initiation complex occurs at the TATA box or the initiator (Matsui et al., 1980; Sawadogo and Roeder, 1985; Buratowski et al., 1989). In addition to RNA polymerase II (RNApol II), the basal transcription factors that are required for transcription initiation are TFIIA, -B, -D, -E, -F and -H. These were identified in nuclear extracts that can support basal transcription
Activated transcription
Assembly of the basal transcriptional machinery on prom oter elements is usually not sufficient to direct transcription of a gene. Thus, in addition to the regulatory elements located in the prom oter region of genes, other DNA sequences situated upstream or downstream of the transcriptional initation site are req u ired for activated and tissue specific expression. These are referred to as enhancers and silencers. They usually consist of m ultiple cis- acting elements w ith which sequence specific transcription factors interact. Most transcription factors contain a DNA binding dom ain and a separable activator/ repressor dom ain (Mitchell and Tjian, 1989) and there is no strict correlation betw een function (enhancer/supressor) of the regulatory dom ain and amino acid composition. However, activator dom ains are often acidic, glutam ine or proline rich whilst repressor dom ains have been found to be ala nine rich or basic (Han and Manley, 1993; Saha et al., 1993; Licht et al., 1994).
Enhancers
Enhancers and silencers can be located 5' or 3' of the start site and are often kilobases aw ay from the prom oter of the linked gene (Banerji et al., 1981). Enhancers are cis-acting DNA elements that increase the rate of tran scription of nearby genes in an orientation and relatively distance indepen dent m anner (Banerji et al., 1981; Moreau et al., 1981; Serfling et al., 1985; Treisman and Maniatis, 1985; Weber and Schaffner, 1985; Ptashne, 1986).
prin-ciple is exemplified by the m urine CD4 enhancer and is described in detail below.
The CD4 enhancer
The CD4 enhancer is located 13kb upstream of the transcriptional initi ation site of the m urine CD4 gene. It forms a T cell specific DNasel-HSS in the CD4 gene locus and functions in an orientation and position independent m anner w hen transfected into a Jurkat T cell line. Deletion analysis has local ized the m inimal enhancer to a 339bp fragment (Sawada and Littman, 1991). In transgenic mice the CD4 enhancer is constitutively active in all T cells and directs expression of linked transgenes in all thymocytes and T cells (Sawada et al., 1994; Siu et al., 1994; Salmon et al., 1996).
In vitro D N asel footprinting identified three protected sites (CD4-1, CD4-2 and CD4-3), one of w hich (CD4-2) is T cell specific (Saw ada and Littman, 1991). The T cell specific site contains a sequence closely related to nuclear protein binding sites w ithin enhancers of other T cell specific genes such as Ta2 (Ho et al., 1989), CD3-e (van de Wetering et al., 1991) and CD2-E5 (Lake et al., 1990). CD4-2 contains a sequence th at b in d s T C F-la/L E F -1 (Waterman et al., 1991), a factor expressed in T and pre-B cell lines b u t not m ature B cells (Travis et al., 1991). The other two sites (CD4-1 and CD4-3) contain binding sites for bHLH proteins (Weintraub et al., 1991) and addition ally have AP-1 and AP-2 binding sites. Point m utations in the DNasel pro tected sites CD4-1 and CD4-2 reduce the activity of the enhancer by two to four fold whilst m utations in CD4-3 results in a decrease of enhancer function of up to 90% (Sawada and Littman, 1991).
M echanism s of enhancer function
tw o p ro m o ters w ere p laced adjacent to th e SV40 enhancer, p referen tial enhancem ent of the proximal prom oter could be observed (de Villiers et al., 1982; Wasylyk et al., 1983; Kadesch and Berg, 1986). Based on this observa tion it was proposed that the enhancer w orks as an entry site for transcription factors which then slide along the DNA. However, it is more likely that the preferential activation of the proximal prom oter was due to a distance effect
(Wasylyk et al., 1984).
The structural model proposes that the surrounding chrom atin struc ture is changed by enhancers and that this allows access of transcription fac tors. However, experiments in which enhancer sequences were topologically separated from the prom oter of the reporter gene argue against the sliding and remodelling models (Plon and Wang, 1986; Mueller-Storm et al., 1989).
The prevailing model of enhancer function is that of protein - protein interactions. Thus, enhancers exert their influence by acting as binding sites for transcription factors which subsequently interact w ith prom oter bound factors of the transcriptional machinery. Transcription factors have been sh o w n to often contain DN A b in d in g d o m ain s th a t are in su fficien t for enhancer function and activation domains which can interact w ith other fac tors (Ma and Ptashne, 1987; Ptashne, 1988). Transcriptional activators or repressors that bind at enhancers or silencers respectively are thought to con tact components of the basal transcriptional machinery either directly or, indi rectly via interm ediatory proteins. Thus, and as m entioned above, they could interact directly w ith prom oter factors such as RNApol II (Allison and Ingles, 1989; Brandi and Struhl, 1989), the TATA box binding factor TFIID (Hai et al., 1988; Horikoshi et al., 1988) and other prom oter elements such as the CAAT box binding proteins (Antoniou and Grosveld, 1990) or the transcription fac tor SPl (Takahashi et al., 1986).
at the promoter. Recent work that examined transcription of tem plates at the single cell level has led to the formation of the probability m odel for enhancer function. This states that an enhancer acts to increase the probability of a par ticular tem plate to be transcribed rather than to increase the num ber of tran scribing RNA polym erase complexes on a particular tem plate (Weintraub, 1988; Walters et al., 1995).
Silencers
In contrast to enhancers which exert a positive role on transcription, silencers decrease the rate of, or prevent transcription from a promoter. Such negative regulatory elements have been described for a range of genes such as the im m unoglobulin genes (Kadesch et al., 1986; Imler et al., 1987), AFP gene (Camper and Tilghman, 1989), ovalbumin prom oter (Gaub et al., 1987), a-in terfe ro n p ro m o ter (Kuhl et al., 1987) an d the p-interferon p ro m o te r (G oodboum et al., 1986; Zinn and Maniatis, 1986).
Whilst the specific repression of genes is usually due to the loss of the function of an activator protein, the action of sequence specific transcriptional repressors w ould be to prevent prom oter access by transcriptional activators as is the case for c-myc (Amati et al., 1992; Kretzner et al., 1992a; Kretzner et al., 1992b) or steric occlusion of activator binding sites (G oodbourn, 1990; Clark and Docherty, 1993; Herschbach and Johnson, 1993). Repressors can also m ediate their effect by interfering w ith the activity or assembly of the basal transcriptional machinery (Jaynes and O'Farrell, 1991; Baniahmad et al., 1992; H an and Manley, 1993) or reduce the num ber of functional pre-initation complexes that form on the prom oter (Johnson and Krasnow, 1992).
enhancer function since removal of these sequences allows enhancer m ediat ed transcription in fibroblasts (Imler et al., 1987). These elements have been show n to be bound by a factor, NF-|iNR, that is present in cells that do not express the Ig heavy chain (Scheuermann and Chen, 1989). Similarly, and as described below, the CD4 silencer overrides the activity of the CD4 enhancer in the CD4"CD8' DN thymocytes and CD4"CD8‘^ T cells (Sawada et al., 1994; Siu et al., 1994).
The CD4 silencer
DNasel-HSS m apping around the m urine CD4 gene identified further DNasel-HSS separate from the enhancer (described above), one of which is located w ithin the first intron on the CD4 gene (Sawada et al., 1994; Siu et al., 1994). Inclusion of this DNasel-HSS site in transgene constructs resulted in subset specific, developm entally regulated expression of both HLA-B7 and hCD2 reporter gene constructs in transgenic mice (Sawada et al., 1994; Siu et al., 1994). The silencer is position and orientation independent and functions in CD4"CD8" DN thymocytes, CD4"CD8'*' SP T cells, B cells and macrophages. It is, however, inactive in the CD4"'"CD8'‘' DP thym ocytes and is therefore developm entally regulated (Sawada et al., 1994; Siu et al., 1994).
fied binding regions results in abolition of silencer function and expression of th e re p o rte r tran sg en e in in a p p ro p ria te cell su b sets in tran sg en ic m ice (Duncan et al., 1996).
Locus Control Regions
DNA constructs that contain enhancer motifs have been used to gener ate transgenic mice. Even though these constructs were active w hen trans fected into cell lines, they were found to be often silent in the transgenic mice. In addition, the expression level of the linked gene did not correlate w ith the copy num ber integrated into the genome. These effects were attributed to the site of transgene integration into the genome. This has led to the identifica tion of regulatory elements that can overcome the repressive effects of chro m atin (see below) and have been term ed locus control regions (LCR).
In the case of the p globin locus several erythroid specific DNasel-HSS have been m apped betw een 6kb to 18kb upstream of the 5' e-globin gene (Tuan et al., 1985; Forrester et al., 1986; Grosveld et al., 1987). Deletion of these sites results in formation of inactive chromatin in the locus and in yp thalassaem ia (van der Ploeg et al., 1980; Kioussis et al., 1983). Linkage of these sites to the p globin gene results in copy num ber dependent, position independent transgene expression in transgenic mice (Grosveld et al., 1987).
both erythroid specific and ubiquitous nuclear proteins (Philipsen et al., 1990; Talbot et al., 1990).
Chromatin Structure And Gene Regulation
The interaction of the transcriptional m achinery w ith the various DNA binding sites described above does not take into account the compaction of nucleosomal DNA and the steric constraints imposed on it by nucleosomal packaging of DNA and higher order structure. There is now extensive exper imental evidence for the interaction between transcription factors and histone proteins in the regulation of gene expression (Croston and Kadonaga, 1993; Svaren and Horz, 1993). For example, m utations in histone genes have major effects on transcriptional activation and repression (Grunstein, 1990). Indeed, the generally repressive nature of chromatin structure has long been appreci ated in transcriptional regulation but can also facilitate the activation of spe cific genes (Wasylyk and Chambon, 1979; Grunstein, 1990; Faranjape et al., 1994). This p art of the introduction therefore focuses on the packaging of DNA into nucleosomal structures and the role that this condensation has in respect to gene regulation.
H istones and nucleosom e structure
D igestion of chromosomal DNA preparations w ith enzym es such as micrococcal nuclease has shown that eukaryotic DNA is organized into a reg ularly repeating protein-D N A complex called the nucleosom e (Kornberg, 1977). The basic unit of chromatin is the nucleosome, a complex of DNA and proteins called histones. Genomic DNA is w ound around a core of histone proteins to give, w hen viewed in the electron microscope, a bead-on-a-string appearance (Kornberg, 1974; Kornberg and Thomas, 1974; Olins and Olins, 1974).
contain a globular dom ain through which histone-histone and histone-DNA interactions occur. X-ray crystallographic data have show n that each histone consists of a structured three-helix dom ain, also called the histone fold, as well as two unstructured tails. The amino-terminal tails of the core histones contain a large num ber of lysine and arginine amino acid residues and are modified by acétylation which can have im portant consequences for the inter action of transcription factors w ith their recognition sites in chrom atin (Lee et al., 1993). In addition to the four core histones most eukaryotic cells contain the linker histone H I that is also highly basic in amino acid composition, con tains a central globular dom ain and is slightly larger than the core histones (>20 kDa) (van Holde, 1988; Hayes et al., 1991; Wolffe, 1992).
microscopy, sedim entation and nuclease digestion studies support the loop b a sed m o d el (Benyajati an d W orcel, 1976; C asser an d L aem m li, 1987). Indeed, a nuclear m atrix or scaffold that fastens the loops can be isolated (Mirkovitch et al., 1984) and scaffold or matrix attachm ent regions (SAR or MAR) several hundred base pairs in size rich in A /T base composition specif ically bind to interphase or m etaphase scaffolds (M irkovitch et al., 1984; Mirkovitch et al., 1988). SARs were first identified in Drosophila melanogaster
and co-localized w ith functional gene regulatory elements such as prom oters and enhancers (Mirkovitch et al., 1984; Casser and Laemmli, 1986) and since then have also been identified in mammals such as in the im m unoglobulin |i- heavy and K-light chain enhancers (Cockerill and C arrard, 1986a; Cockerill et al., 1987). DNA sequences found within SARs include recognition sites for topoisom erase II (Cockerill and C arrard, 1986b; C asser et al., 1986). This enzyme has been localized to the bases of chromatin loops (Earnshaw et al., 1985; Earnshaw and Heck, 1985) and show n to bind SAR sequences (Adachi et al., 1989). The function of topoisomerase II m ay be to regulate the superhe lical torsion in a loop which m ay occur due to transcriptional activity.
The compartmentalization of DNA into such Toop" dom ains has given rise to a model of the chromatin dom ain as a functional regulatory unit. In other w ords, a chrom osom al dom ain form s a u n it of gene expression in w hich activated transcription from a prom oter located w ithin the dom ain results only from the action of a combination of enhancers/silencers w ithin the same domain. This w ould thereby prevent the enhancer/silencer combi nation from acting on other genes located outside of the domain.
C hrom atin structure and transcription
P H 0 5 gene indicating that nucleosomes can act as transcriptional repressors (Han and Grunstein, 1988; H an et al., 1988). Furthermore, genetic analyses of transcriptional processes in yeast have identified several genes th at have pleiotropic effects. It was found for example that common genes are required for transcription of genes belonging to the sucrose metabolism pathw ay or the endonuclease gene which is involved in m ating control (Abrams et al., 1986; Laurent et al., 1991; Peterson and H erskow itz, 1992). The required genes are term ed SWI/SNF and biochemical evidence now indicates that they form a large 'a c tiv a to r' com plex th at does n o t bin d DNA directly b u t is thought to destabilize nucleosomes (Tamkun, 1995). In vitro this complex allows transcription factors to bind their cognate recognition sites on reconsti tuted chromatin templates (Cote et al., 1994; Imbalzano et al., 1994).
In addition, analysis of prom oter regions such as those of the yeast P H 0 5 gene and the mouse m am m ary tum our virus (MMTV) promoter, locat ed in the viral long terminal repeat (LTR), has substantiated the observations m ade from the genetic analysis m ade in yeast and described above. The LTR is incorporated into six nucleosomes (Richard-Foy and Hager, 1987) that have a negative effect on transcription since they prevent the basal transcriptional machinery to assemble at the prom oter (Archer et al., 1992). Similarly regula tion of transcription at the PH 05 prom oter involves the m asking two binding sites for tran scrip tio n factors (Aimer and H orz, 1986; A im er et al., 1986; Fascher et al., 1990). Changes in DNA sequence in adjacent nucleosomes can influence chrom atin remodelling at these prom oters suggesting that precise organization of the nucleosomal structure is essential for correct transcrip tional regulation (Fascher et al., 1990; Straka and Horz, 1991).
m oter hsp26 is an example (Thomas and Elgin, 1988). Four binding sites for the heat shock transcription factor HSTF are present in the promoter, two of w hich are incorporated into nucleosom es and are inaccessible to the tran scription factor (Taylor et al., 1991). Incorporation of the DNA into nucleoso mal structures brings the other two binding sites, located in the linker DNA regions close together and thereby m ay facilitate transcription (Thomas and Elgin, 1988). In a similar situation, the positioning of a nucleosome betw een the prom oter and enhancer of the Xenopus laevis vitellogenin gene (Jackson and Benyajati, 1993; Schild et al., 1993) brings the binding sites for the oestro gen receptor and the N Fl transcription factor into close proximity enhancing transcription up to ten fold (Schild et al., 1993).
D N asel hypersensitive sites
Distortions of chromatin structure of active areas can often be detected by an increase in sensitivity to nucleases such as DNasel. The increase in sen sitivity to DNasel digestion in expressing tissues was first noted for the glo bin and ovalbum in genes (Garel and Axel, 1976; W eintraub and Groudine,
1976) and sensitivity was specific for the active loci of the genes. It was there fore proposed that these genes have an altered chromatin conformation. This type of DNasel sensitivity has been attributed to histone acétylation (Vidali et al., 1978), absence of histone H I (Huang and Cole, 1984; Karpov et al., 1984; Schlissel and Brown, 1984) and underm ethylation of DNA sequences (Tazi and Bird, 1990).
Control Of CD8 Gene Expression
Based on DNA sequence analysis of the genomic 5' and 3' flanking sequences of the CD8a/Lyt-2 and CD8p/Lyt-3 a search for DNA regulatory sequences has been carried out by several groups.
CDSa gene regulatory elem ents
Nakauchi et al. (1987) have carried out sequence analysis of the genom ic H indlll fragm ent th at has been show n to encode the C D 8a/L yt-2 gene. Using prim er extension and SI nuclease m apping techniques the transcrip tional initiation site was determined to lie 334nt - 335nt upstream of the trans lational initiation codon ATG. Based on these findings a TATA box like sequence (sequence of TATTAA) was found to be located 29bp upstream of the transcriptional initiation site. No further ATG initiation codons or other TATA box like sequences were found to lie between the m apped initiation site and the methionine start codon of the CD8a leader peptide (Nakauchi et al., 1987).
In addition, two further groups have investigated potential regulatory elem ents located upstream of the C D 8a/L yt-2 gene. L andry et al. (1993) employed DNasel-HSS m apping to identify three DNasel-HSS located at the prom oter region, at -2kb and -4kb upstream of the C D 8a/Lyt-2 gene. The DNasel-HSS were found to be restricted to cell lines which express CD8a and were absent from CD4 single positive or CD4"CD8" DN cell lines. One of th ese D N asel-H SS sites, located at -4kb w as fu rth e r an aly ze d b y elec trophoretic mobility shift assays. Two subfragments isolated from the region encompassing this DNasel-HSS were found to form sequence specific DNA- protein complexes w ith extracts obtained from CD8'*'CD4''‘ and CD8"CD4"^ cell lines b u t not with B cell or non-lymphoid HeLa nuclear extracts. Cross competition experiments between the two fragments indicate that both bind the sam e transcription factor. M éthylation interference analysis identified tw o sequence elements (GATTAATGA and AGATAG) that bear significant hom ology to sequences in the hum an p-globin enhancer (Wall et al., 1988) and the Ay-globin prom oter (Martin et al., 1989). Using recombinant GATA-3, a T lymphocyte restricted transcription factor (Joulin et al., 1991) the -4kb DNasel-HSS was show n to contain a functional GATA-3 binding site.
