Evaluation of the LacO/LacR Tethering Method Coupled to a High Content Analysis: Study of the in vivo Interaction Between the NuRD Complex and the Transcription Factor MITF in Human Melanoma
(2017) MOBM01 20171Degree Projects in Molecular Biology
- Abstract
- Mammalian cells are frequently under stress leading to DNA damage because of UV exposure, ionizing radiation or genotoxic chemicals. Cells have evolved a DNA damage response (DDR) mechanism to ensure survival and pass on the correct genetic information to the next generation. MITF is a melanocyte-specific transcription factor. It has been shown to accumulate in the vicinity of DNA damage and its expression is associated with a stronger activation of the DDR. During DNA damage, the NuRD complex also accumulates in the damaged region and contributes to the repair via its chromatin remodelling and histone deacetylation activity. Preliminary results suggested that MITF may interact with the NuRD complex. We then hypothesized that the effect of... (More)
- Mammalian cells are frequently under stress leading to DNA damage because of UV exposure, ionizing radiation or genotoxic chemicals. Cells have evolved a DNA damage response (DDR) mechanism to ensure survival and pass on the correct genetic information to the next generation. MITF is a melanocyte-specific transcription factor. It has been shown to accumulate in the vicinity of DNA damage and its expression is associated with a stronger activation of the DDR. During DNA damage, the NuRD complex also accumulates in the damaged region and contributes to the repair via its chromatin remodelling and histone deacetylation activity. Preliminary results suggested that MITF may interact with the NuRD complex. We then hypothesized that the effect of MITF during the DDR may result from the recruitment of the NuRD complex at the site of DNA damage that would create a localized decondensation of the chromatin and ease the development of the response. In this report, we investigated the in vivo interaction between NuRD and MITF using the novel LacO/LacR tethering system coupled to High Content Analysis. After validating the protocol, we confirmed that MITF can recruit some, but not all of the NuRD complex factors. This interaction was linked to the N-terminal part of MITF, although the mechanism of recruitment remain unclear. Overall, we could confirm previously obtained results and provided additonal insights into how MITF might contribute to chromatin regulation during the DNA damage response. (Less)
- Popular Abstract
- MITF-NuRD complex interaction enhances DNA Damage Response
Exposure to UV, ionizing radiation or genotoxic chemicals produce frequently stress into mammalian cells leading to DNA damage and genome instability. Multiple DNA Damage Response pathways have evolved in order to maintain survival and the right pass to genetic information to future generations. Here we described using the LacO/LacR tethering system coupled to a high content system, the potential in vivo interaction in between two factors (MITF and NuRD complex) involved in the DNA Damage Repair mechanism and the possible enhancement of DNA Damage Repair due to their interaction.
MITF is a melanocyte-specific transcription factor and it has been shown to accumulate in DNA... (More) - MITF-NuRD complex interaction enhances DNA Damage Response
Exposure to UV, ionizing radiation or genotoxic chemicals produce frequently stress into mammalian cells leading to DNA damage and genome instability. Multiple DNA Damage Response pathways have evolved in order to maintain survival and the right pass to genetic information to future generations. Here we described using the LacO/LacR tethering system coupled to a high content system, the potential in vivo interaction in between two factors (MITF and NuRD complex) involved in the DNA Damage Repair mechanism and the possible enhancement of DNA Damage Repair due to their interaction.
MITF is a melanocyte-specific transcription factor and it has been shown to accumulate in DNA damage areas. The NuRD complex is a chromatin remodeling and histone deacetylation factor and it accumulates at the vicinity of DNA damage regions. We hypothesized then, that MITF may recruit NuRD complex into DNA damage sites and due to its decondensation of chromatin activity will enhance the MITF action in DNA Damage Repair.
We tested specificity of LacO/LacR tethering system to recognize protein interaction taking advantage of the previously known dimerization of MITF capability. We determine that the method was able to determine interaction between MITF. We went further and detailed the specific binding area involved in MITF dimerization concluding that the domain containing the bHLH-LZ is involved in MITF dimerization.
Finally, we tested the MITF-NuRD interaction by co-transfecting MITF with GATAD2B, MTA1 and MBD2, which are specific NuRD subunits. In this case, we determine the positive interaction between MITF and NuRD complex as accumulation of both could be seen in the same region (Figure 1). We also studied more in depth the specific are of interaction and we conclude that MITF
and NuRD are interacting at the 1-180 position of MITF.
We conclude that the MITF-NuRD interact. Therefore, a better understanding of the regulation of interaction and its role in DNA Damage Repair will lead to potential therapeutic implications.
Advisor: Dr. Romuald Binet and Prof. Colin R. Goding NDM Ludwig Institute for Cancer Research, Oxford University
Master’s degree Project in Molecular Biology 30 credits 2017
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8925406
- author
- Lopera Burgueño, Raquel
- supervisor
- organization
- course
- MOBM01 20171
- year
- 2017
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 8925406
- date added to LUP
- 2017-09-12 09:38:34
- date last changed
- 2017-09-12 09:38:34
@misc{8925406,
abstract = {{Mammalian cells are frequently under stress leading to DNA damage because of UV exposure, ionizing radiation or genotoxic chemicals. Cells have evolved a DNA damage response (DDR) mechanism to ensure survival and pass on the correct genetic information to the next generation. MITF is a melanocyte-specific transcription factor. It has been shown to accumulate in the vicinity of DNA damage and its expression is associated with a stronger activation of the DDR. During DNA damage, the NuRD complex also accumulates in the damaged region and contributes to the repair via its chromatin remodelling and histone deacetylation activity. Preliminary results suggested that MITF may interact with the NuRD complex. We then hypothesized that the effect of MITF during the DDR may result from the recruitment of the NuRD complex at the site of DNA damage that would create a localized decondensation of the chromatin and ease the development of the response. In this report, we investigated the in vivo interaction between NuRD and MITF using the novel LacO/LacR tethering system coupled to High Content Analysis. After validating the protocol, we confirmed that MITF can recruit some, but not all of the NuRD complex factors. This interaction was linked to the N-terminal part of MITF, although the mechanism of recruitment remain unclear. Overall, we could confirm previously obtained results and provided additonal insights into how MITF might contribute to chromatin regulation during the DNA damage response.}},
author = {{Lopera Burgueño, Raquel}},
language = {{eng}},
note = {{Student Paper}},
title = {{Evaluation of the LacO/LacR Tethering Method Coupled to a High Content Analysis: Study of the in vivo Interaction Between the NuRD Complex and the Transcription Factor MITF in Human Melanoma}},
year = {{2017}},
}