11-9-08_ppt
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INTRODUCTION Introduction
The human cancer genome, or oncogenome harbors numerous alterations at the level of the chromosomes, the chromatin (the fibres that constitute the chromosomes), and the nucleotides. These alterations include irreversible aberrations in the DNA sequence or structure and in the number of particular sequences genes or chromosomes, (that is the copy number of the DNA). They also include potentially reversible changes known as epigenetic modifications to the DNA and/ or to the histone proteins, which are closely associated with the DNA in chromatin. These reversible, and irreversible changes can affect hundreds to thousands of gene and/or regulatory transcripts. Collectively they result in the activation and inhibition of various biological events thereby causing aspects of cancer pathophysiology including angiogenesis, immune
GENOMIC AND EPIGENOMIC ABERRATIONS IN CANCER
DETECTING CANCER BIOMARKERS
In this work we report for the first time an in silico approach for the detection and screening of genes which might be upregulated in cancer. The approach involves the identification and analyses of regulatory motif binding sites in the genes implicated in various human carcinomas and correlating them with the reported expression patterns of the genes.
ComA Positive regulator of genes involved in late-growth expression and in response to environmental stress; is phosphorylated by ComP; may cause DNA bending by bridging two binding sites
RocR NtrC/NifA transcriptional activator (cf. LevR). Sigma 54-dependent activators generally bind two inverted repeat sequences called UAS, which are located approx. 100bp upstream from the -12/-24 promoters. DNA bending is used for activation (AhrC may be involved). Inducible by ornithine or citrulline? At least upstream UAS1 is the target of RocR.
AbrB Bi-functional; expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation; acts as the Spo0A-AbrB circuit; also involved in catabolite repression
CcpA Also called AlsA; common repressor in catabolite repression (CR) but may act as a positive regulator of genes involved in excretion of excess carbon; its binding site is called CRE; it binds to fructose-1,6-bisphosphate and HPr.
TnrA Positively regulates many genes for degrading nitrogen-containing compounds but negatively regulates glnR. Binds to the same sequence with GlnR (repressor).
Xre
Repressor of a phage-like bacteriocin, PBSX (phibacin damaged-prophage). Helix-turn-helix protein. Acts at the operator-promoter region.
GlnR Originally identified as a repressor of glnRA operon; binds to the same sequence with TnrA (activator).
DegU
Pleiotropic regulator involved in various post-exponential phase responses; makes a two component system with DegS kinase
CtsR Binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAANANA/GGTCAAA); three functional domains: HTH DNA-binding, dimerization, and putative heat-sensing domains; active as a dimer; specifically degraded by ClpP and ClpX at 37 degrees C; labile and substrate of the ClpCP protease under stress conditions (autoregulation by controlled proteolysis); negatively regulates its own synthesis.
HrcA
The promoters of class I heat-inducible genes are sigA-dependent and have an inverted repeat, called the CIRCE (controlling IR of chaperone expression) element, which is highly conserved among eubacteria; heat-shock and general stress responses are reviewed in Hecker, M. et al., 1996.
PerR Transcription induced by hydrogen peroxide, by general stress, or by entry into stationary phase under conditions of iron and manganese limitation (class III stress response); expression modulated by Ca2+; mutants lead to higher expression from all peroxide regulon promoters (mrgA, katA, hemAXCDBL, and ahpCF), but has no effect on spore resistance to alkyl hydroperoxides, heat or hydrogen peroxide.
PucR Regulation of puc genes (purine degradation).
GerE
Positive(?) regulator which affects transcription of many genes in the mother cell during the late stages of sporulation.
MntR Regulation of manganese transport (repression of mntH in high Mn(II) conditions, activation of mntABCD under low Mn(II) conditions).
GltR
It activates the transcription of gltAB in the absense of the normal regulator, GltC. It also negatively regulates its own expression. cf. GltC.
FNR Same binding consensus with E. coli CAP exists in the narK-fnr operon.
AhrC
Novel mode of DNA recognition; represses the arginine biosynthesis genes and activates the arginine catabolism genes; positive role of AhrC may involve proteinprotein interaction with RocR.
Spo0A A key bi-functional regulator to control developmental transcription activities. Increases its affinity after phosphorylation (phosphorelay system). Spo0F is required for the phosphorylation. Two-domain structure. Binding consensus is called 0A box and can be located downstream of the initiation site. Often, two adjacent boxes are found. These listed sites might be viewed in its complementary strand.
ResD
ResD seems to form a two-component signal transduction system with resE and plays a regulatory role in respiration. Interactions with resABCDE operon and ctaA may be indirect.
Mta
The N-terminal domain of Mta(MtaN) acts as a constitutive activator of the transcription of bmr and blt genes.
Fur
PurR A purine repressor which mediates adenine nucleotide-dependent regulation of pur operon. GAAC-N24-GTTC motif seems necessary for its binding but this motif was not required for its binding to purA.
SinR Dual-function regulator which is essential for the late-growth processes of competence and motility and is also a repressor of others, e.g., sporulation and subtilisin synthesis. Might be a leucine zipper protein. In aprE there are two binding sites and SinR binds more strongly to the distal site, which contains two dyad symmetry sites.
Negative regulation of siderophore biosynthesis and transcription of ferri-siderophore uptake genes.
AraR Transcriptional regulator (LacI family); negative regulation of the L-arabinose metabolic operon (araABDLMNPQ); alternate gene name: araC, yvbS.
LevR
GltC
Like many members of LysR family, GltC is encoded just upstream of and in the opposite orientation to its target genes (gltAB); it negatively autoregulates itself. The binding site for the autoregulation is the same with the box I of gltA (-71:-57) in its genome position. Box II of gltA overlaps with its -35 region. cf. GltR.
CodY
Transcriptional regulator; negative regulation of srfA and comK genes (in the presence of casamino-acids), dpp operon.
Iolr A negative regulator (presumably a repressor) which exists immediately upstream of the iol operon in the opposite direction.
BkdR
Activator which regulates the neighboring operon. Note that -144:-130 and -108:-120 makes a palindromic structure.
SpoiiiD
A bi-functional transcription factor which regulates temporal expression of many genes in the mother cell as well as GerE.
Zur
Zinc-specific repression of operons implicated in zinc uptake (yciC, ycdHIyceA).0
ComK
The results indicate that the genes reported to be upregulated in cancer possess a specific pattern of regulatory motif binding sites and future cancer biomarkers can be screened and tested with the presence of the same set of regulatory motif binding sites. A correspondence analysis was also performed along with a correlation analysis to further establish our findings. This approach could prove to be beneficial for screening of the specific genes before workers embark upon wet lab experiments.
Selected Genes (on the basis of Correspondence Analysis)
CORRESPONDENCE PLOT CorrespondencePlot 14
DIM(2)
7
1.3 3 4 6 76 0 2 3 5 3.3 7 18
0
83 9 1 2 3 4 5 6 7 7 .6 2 9 0 1 4 8 5 0 5 1 4 9 3 6 2 7 8 .9 32 8 5 6 7 1 4 2 5 7 1 4 3 6 8
-7 1.23 2
-14 -14
-7
0
D IM (1)
7
14
Transcription Factors
SCATTER PLOT MATRIX
Total number of Genes
We wish to develop a tool for the analysis and screening of the possible cancer gene sequences, thus making wet lab experiments more specific, which would be beneficial for controlling the deadly disease. We provide an overview of gene expression analyses and upregulation, in case of human oncogene, and suggest future uses of transcription regulatory analyses to rationalise the observations made through computational
FUTURE PROSPECT Contd… We can construct a large number of different profile matrices from a given sample, by varying the starting positions, and may grade them against the predicted set of motifs found from already determined human oncogenes. Based on this grading and on different principles. We can formulate the human cancer biomarker finding problem to give efficient algorithm. The algorithms can be integrated to design a "Human Cancer Biomarker Finding Tool". Thus users can submit, nucleotide sequences of novel genes, to this tool in order to find patterns/ motifs related to human oncogenes.
FUTURE PROSPECT Contd ….. Improvement in cancer classifications have
been central to advances in cancer treatment. Although the distinction between different forms of cancer has been well established , it is still not possible to establish a clinical diagnosis on the basis of a single test. In a recent study, acute myeloid leukaemia and acute lymphoblastic leukaemia were successfully distinguished based on the expression profiles of these cells. Thus the human oncogene expression profiles may provide a generic strategy for classifying all
The human cancer genome, or oncogenome harbors numerous alterations at the level of the chromosomes, the chromatin (the fibres that constitute the chromosomes), and the nucleotides. These alterations include irreversible aberrations in the DNA sequence or structure and in the number of particular sequences genes or chromosomes, (that is the copy number of the DNA). They also include potentially reversible changes known as epigenetic modifications to the DNA and/ or to the histone proteins, which are closely associated with the DNA in chromatin. These reversible, and irreversible changes can affect hundreds to thousands of gene and/or regulatory transcripts. Collectively they result in the activation and inhibition of various biological events thereby causing aspects of cancer pathophysiology including angiogenesis, immune
GENOMIC AND EPIGENOMIC ABERRATIONS IN CANCER
DETECTING CANCER BIOMARKERS
In this work we report for the first time an in silico approach for the detection and screening of genes which might be upregulated in cancer. The approach involves the identification and analyses of regulatory motif binding sites in the genes implicated in various human carcinomas and correlating them with the reported expression patterns of the genes.
ComA Positive regulator of genes involved in late-growth expression and in response to environmental stress; is phosphorylated by ComP; may cause DNA bending by bridging two binding sites
RocR NtrC/NifA transcriptional activator (cf. LevR). Sigma 54-dependent activators generally bind two inverted repeat sequences called UAS, which are located approx. 100bp upstream from the -12/-24 promoters. DNA bending is used for activation (AhrC may be involved). Inducible by ornithine or citrulline? At least upstream UAS1 is the target of RocR.
AbrB Bi-functional; expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation; acts as the Spo0A-AbrB circuit; also involved in catabolite repression
CcpA Also called AlsA; common repressor in catabolite repression (CR) but may act as a positive regulator of genes involved in excretion of excess carbon; its binding site is called CRE; it binds to fructose-1,6-bisphosphate and HPr.
TnrA Positively regulates many genes for degrading nitrogen-containing compounds but negatively regulates glnR. Binds to the same sequence with GlnR (repressor).
Xre
Repressor of a phage-like bacteriocin, PBSX (phibacin damaged-prophage). Helix-turn-helix protein. Acts at the operator-promoter region.
GlnR Originally identified as a repressor of glnRA operon; binds to the same sequence with TnrA (activator).
DegU
Pleiotropic regulator involved in various post-exponential phase responses; makes a two component system with DegS kinase
CtsR Binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAANANA/GGTCAAA); three functional domains: HTH DNA-binding, dimerization, and putative heat-sensing domains; active as a dimer; specifically degraded by ClpP and ClpX at 37 degrees C; labile and substrate of the ClpCP protease under stress conditions (autoregulation by controlled proteolysis); negatively regulates its own synthesis.
HrcA
The promoters of class I heat-inducible genes are sigA-dependent and have an inverted repeat, called the CIRCE (controlling IR of chaperone expression) element, which is highly conserved among eubacteria; heat-shock and general stress responses are reviewed in Hecker, M. et al., 1996.
PerR Transcription induced by hydrogen peroxide, by general stress, or by entry into stationary phase under conditions of iron and manganese limitation (class III stress response); expression modulated by Ca2+; mutants lead to higher expression from all peroxide regulon promoters (mrgA, katA, hemAXCDBL, and ahpCF), but has no effect on spore resistance to alkyl hydroperoxides, heat or hydrogen peroxide.
PucR Regulation of puc genes (purine degradation).
GerE
Positive(?) regulator which affects transcription of many genes in the mother cell during the late stages of sporulation.
MntR Regulation of manganese transport (repression of mntH in high Mn(II) conditions, activation of mntABCD under low Mn(II) conditions).
GltR
It activates the transcription of gltAB in the absense of the normal regulator, GltC. It also negatively regulates its own expression. cf. GltC.
FNR Same binding consensus with E. coli CAP exists in the narK-fnr operon.
AhrC
Novel mode of DNA recognition; represses the arginine biosynthesis genes and activates the arginine catabolism genes; positive role of AhrC may involve proteinprotein interaction with RocR.
Spo0A A key bi-functional regulator to control developmental transcription activities. Increases its affinity after phosphorylation (phosphorelay system). Spo0F is required for the phosphorylation. Two-domain structure. Binding consensus is called 0A box and can be located downstream of the initiation site. Often, two adjacent boxes are found. These listed sites might be viewed in its complementary strand.
ResD
ResD seems to form a two-component signal transduction system with resE and plays a regulatory role in respiration. Interactions with resABCDE operon and ctaA may be indirect.
Mta
The N-terminal domain of Mta(MtaN) acts as a constitutive activator of the transcription of bmr and blt genes.
Fur
PurR A purine repressor which mediates adenine nucleotide-dependent regulation of pur operon. GAAC-N24-GTTC motif seems necessary for its binding but this motif was not required for its binding to purA.
SinR Dual-function regulator which is essential for the late-growth processes of competence and motility and is also a repressor of others, e.g., sporulation and subtilisin synthesis. Might be a leucine zipper protein. In aprE there are two binding sites and SinR binds more strongly to the distal site, which contains two dyad symmetry sites.
Negative regulation of siderophore biosynthesis and transcription of ferri-siderophore uptake genes.
AraR Transcriptional regulator (LacI family); negative regulation of the L-arabinose metabolic operon (araABDLMNPQ); alternate gene name: araC, yvbS.
LevR
GltC
Like many members of LysR family, GltC is encoded just upstream of and in the opposite orientation to its target genes (gltAB); it negatively autoregulates itself. The binding site for the autoregulation is the same with the box I of gltA (-71:-57) in its genome position. Box II of gltA overlaps with its -35 region. cf. GltR.
CodY
Transcriptional regulator; negative regulation of srfA and comK genes (in the presence of casamino-acids), dpp operon.
Iolr A negative regulator (presumably a repressor) which exists immediately upstream of the iol operon in the opposite direction.
BkdR
Activator which regulates the neighboring operon. Note that -144:-130 and -108:-120 makes a palindromic structure.
SpoiiiD
A bi-functional transcription factor which regulates temporal expression of many genes in the mother cell as well as GerE.
Zur
Zinc-specific repression of operons implicated in zinc uptake (yciC, ycdHIyceA).0
ComK
The results indicate that the genes reported to be upregulated in cancer possess a specific pattern of regulatory motif binding sites and future cancer biomarkers can be screened and tested with the presence of the same set of regulatory motif binding sites. A correspondence analysis was also performed along with a correlation analysis to further establish our findings. This approach could prove to be beneficial for screening of the specific genes before workers embark upon wet lab experiments.
Selected Genes (on the basis of Correspondence Analysis)
CORRESPONDENCE PLOT CorrespondencePlot 14
DIM(2)
7
1.3 3 4 6 76 0 2 3 5 3.3 7 18
0
83 9 1 2 3 4 5 6 7 7 .6 2 9 0 1 4 8 5 0 5 1 4 9 3 6 2 7 8 .9 32 8 5 6 7 1 4 2 5 7 1 4 3 6 8
-7 1.23 2
-14 -14
-7
0
D IM (1)
7
14
Transcription Factors
SCATTER PLOT MATRIX
Total number of Genes
We wish to develop a tool for the analysis and screening of the possible cancer gene sequences, thus making wet lab experiments more specific, which would be beneficial for controlling the deadly disease. We provide an overview of gene expression analyses and upregulation, in case of human oncogene, and suggest future uses of transcription regulatory analyses to rationalise the observations made through computational
FUTURE PROSPECT Contd… We can construct a large number of different profile matrices from a given sample, by varying the starting positions, and may grade them against the predicted set of motifs found from already determined human oncogenes. Based on this grading and on different principles. We can formulate the human cancer biomarker finding problem to give efficient algorithm. The algorithms can be integrated to design a "Human Cancer Biomarker Finding Tool". Thus users can submit, nucleotide sequences of novel genes, to this tool in order to find patterns/ motifs related to human oncogenes.
FUTURE PROSPECT Contd ….. Improvement in cancer classifications have
been central to advances in cancer treatment. Although the distinction between different forms of cancer has been well established , it is still not possible to establish a clinical diagnosis on the basis of a single test. In a recent study, acute myeloid leukaemia and acute lymphoblastic leukaemia were successfully distinguished based on the expression profiles of these cells. Thus the human oncogene expression profiles may provide a generic strategy for classifying all
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