Analysis of transcription factor binding specificity using ChIP-seq data.
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
Progestin receptor heterogeneity in a breast cancer cell line
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
Isolation of and interaction of nutrients with the linoleoyl-coa desaturase complex
- Authors: Perkins, Denise Mary
- Date: 1990
- Subjects: Cell proliferation , Cancer cells -- Growth -- Regulation , Enzymes -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4558 , http://hdl.handle.net/10962/d1018264
- Description: The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.
- Full Text:
- Date Issued: 1990
- Authors: Perkins, Denise Mary
- Date: 1990
- Subjects: Cell proliferation , Cancer cells -- Growth -- Regulation , Enzymes -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4558 , http://hdl.handle.net/10962/d1018264
- Description: The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.
- Full Text:
- Date Issued: 1990
Zinc inhibition of cell division : its relevance to cancer cells and possible mechanism of action
- Authors: Skeef, Noel Samuel
- Date: 1989
- Subjects: Cell division , Cancer cells -- Growth -- Regulation , Zinc in the body , Zinc -- Physiological effect , Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4144 , http://hdl.handle.net/10962/d1016266
- Description: A description of two techniques used extensively in this study namely cell counting with a "cell counting plate" and argentation TLC for the separation of ω -6 -fatty acids is given. Zn supplementation into GM of two malignant (BL-6 and Hep- 350) and a non-malignant (LLC-MK) cell line/s resulted in an increased uptake of Zn by the cells and progressively suppressed proliferation of particularly the malignant cells. Zn chelation by EDTA suppressed in vitro proliferation of all 3 cell line, this effect being more pronounced in the malignant cells. A dietary Zn deficiency resulted in alopecia in mice and both a dietary Zn deficiency and Zn excess reduced growth of BL-6 tumours implanted subcutaneously in mice. Zn supplementation into GM progressively increased the uptake of [1-¹⁴C]-LA by BL-6 and LLC-MK cells but had a very slight though irregular effect on this parameter in the Hep- 350 cells. Zn supplementation also stimulated desaturase activity in the BL-6 cells. These results suggested that there are select cell lines whose Δ⁶-desaturase activity responds positively to Zn supplementation (e.g. the BL-6 cells). Delta-6-desaturase activity was also assayed in microsome preparations from different tissues. No enzyme activity was detected in the microsomes prepared from the BL-6 tumours. There was no significant effect with the addition of Zn or EDTA, on Δ⁶-desaturase activity of the regenerating liver microsomes. In the resting liver microsomes this enzyme activity was reduced only when EDTA and Zn were added together and when EDTA was added to the reaction medium as well as to the microsome preparations 2 hr before the enzyme activity assay was initiated. The results of these experiments suggested that the Δ⁶-desaturase enzyme in the microsome preparations may have had an adequate amount of Zn with further additions having no stimulatory effect on the enzyme. Two independent mechanisms of control of cell proliferation by low and high Zn are suggested to operate.
- Full Text:
- Date Issued: 1989
- Authors: Skeef, Noel Samuel
- Date: 1989
- Subjects: Cell division , Cancer cells -- Growth -- Regulation , Zinc in the body , Zinc -- Physiological effect , Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4144 , http://hdl.handle.net/10962/d1016266
- Description: A description of two techniques used extensively in this study namely cell counting with a "cell counting plate" and argentation TLC for the separation of ω -6 -fatty acids is given. Zn supplementation into GM of two malignant (BL-6 and Hep- 350) and a non-malignant (LLC-MK) cell line/s resulted in an increased uptake of Zn by the cells and progressively suppressed proliferation of particularly the malignant cells. Zn chelation by EDTA suppressed in vitro proliferation of all 3 cell line, this effect being more pronounced in the malignant cells. A dietary Zn deficiency resulted in alopecia in mice and both a dietary Zn deficiency and Zn excess reduced growth of BL-6 tumours implanted subcutaneously in mice. Zn supplementation into GM progressively increased the uptake of [1-¹⁴C]-LA by BL-6 and LLC-MK cells but had a very slight though irregular effect on this parameter in the Hep- 350 cells. Zn supplementation also stimulated desaturase activity in the BL-6 cells. These results suggested that there are select cell lines whose Δ⁶-desaturase activity responds positively to Zn supplementation (e.g. the BL-6 cells). Delta-6-desaturase activity was also assayed in microsome preparations from different tissues. No enzyme activity was detected in the microsomes prepared from the BL-6 tumours. There was no significant effect with the addition of Zn or EDTA, on Δ⁶-desaturase activity of the regenerating liver microsomes. In the resting liver microsomes this enzyme activity was reduced only when EDTA and Zn were added together and when EDTA was added to the reaction medium as well as to the microsome preparations 2 hr before the enzyme activity assay was initiated. The results of these experiments suggested that the Δ⁶-desaturase enzyme in the microsome preparations may have had an adequate amount of Zn with further additions having no stimulatory effect on the enzyme. Two independent mechanisms of control of cell proliferation by low and high Zn are suggested to operate.
- Full Text:
- Date Issued: 1989
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