YU/BIU Summer 2022 STEM Research Showcase
YU/BIU Summer 2022
SUMMARY:
This research focuses on understanding the properties and interactions of Codanin-1, a protein associated with Congenital Dyserythropoietic Anemia Type I (CDA I). The study investigates the influence of Codanin-1 on the activity and post-translational modification of Asf1, a histone chaperone protein, and explores methods for introducing a degron into the Codanin-1 gene to control protein degradation.
ABSTRACT:
Congenital Dyserythropoietic Anemia Type I (CDA 1); Exploring the Properties and Interactions of Codanin-1
Abigail Razi advised under Dr. Benny Motro and PhD student Moriya Kahta
Congenital Dyserythropoietic Anemia Type I (CDA I) is an autosomal recessive disease associated with abnormalities in erythrocyte precursor proliferation that may be expressed as spongy heterochromatin. The disorder is caused by a mutation in the CDANI gene encoding for the Codanin-1 protein. Codanin-1 is a conserved protein which serves as a protein scaffold and is presumably involved in chromatin assembly and maintenance.
Our research focuses on understanding the function of the Codanin-1 protein. Previously, it was demonstrated that Codanin-1 acts as a protein scaffold to recruit the histone chaperon protein Asf1. Overexpression of Codanin-1 results in a shift in the localization of Asf1 from the nuclear region to the cytoplasm.
In a subsequent analysis of the interactions between Codanin-1 and Asf1, the possible influence of Codanin-1 on post translational modification (PTM) of Asf1 was studied. To do so, HeLa cells underwent an inducible knockout of the gene that codes for Codanin-1. The prediction was that the knockout of this gene would prevent the expression of Codanin-1, thereby influencing the activity and PTM of Asf1. Then, immunoprecipitation was conducted to precipitate Asf1 from treated and untreated cells. Mass spectroscopy was used to identify PTM in Asf1 from cells containing endogenous Codanin-1 and cells containing the inducible knockout of Codanin-1. Differences in PTM between the endogenous Asf1 protein and the Asf1 protein following the inducible knockout of Codanin-1 would not only allude to the influence of Codanin-1 on the activity of Asf1, but more importantly, to larger revelations regarding the role of Codanin-1 in cellular function. The results of this study regarding Asf1 modifications have yet to be identified by mass spectroscopy.
In an additional project, I aimed to change the endogenous Codanin-1 gene by introducing a construct that contained a degron, just prior to its stop codon. A degron is a portion of a protein that is important for controlling protein degradation at the proteasome. In this study, the degron is Auxin (IAA)-dependent, namely in the presence of IAA receptors in the cells. When IAA is added, it binds to its receptor. The receptor recognizes the degron and sends Codanin-1 to the proteasome for degradation.
To insert the plasmid into the genome, a construct was created (referred to as #1217) by adding a 5’ arm and 3’ arm to allow for homologous recombination to occur. Crossover then occurred between the endogenous chromosome and the construct containing the degron and Hygromycin antibiotic resistance. The resulting chromosome contained the same DNA as the endogenous Codanin-1 gene, but also contained the additional degron and antibiotic resistance.
Because there are two chromosomes, it is necessary to introduce the degron into both strands using two constructs that confers cells resistant to both antibiotics: Hygromycin and Blasticidin. The introduction of this gene into the chromosome induces antibiotic resistance by producing a protein that degrades its respective antibiotic, thereby deeming the cell resistant. Since cells will only survive if antibiotic resistance has been introduced, cell survival would indicate successful transformation. In cells where the degron was inserted to both alleles, the addition of Auxin should lead to the degradation of Codanin-1.
Using Crispr methodology, we utilized an sgRNA to cut a strand of chromosomal DNA in order to insert the degron which contained Hygromycin resistance. To insert the degron into the second chromosome, we looked to excise the Hygromycin antibiotic resistance fragment of the plasmid between NheI and NheI and replace it with Blasticidin antibiotic resistance. In order to insert the Blasticidin antibiotic resistant, we edited vector #1217, represented in Figure 1, by inserting a fragment from plasmid #1158 (Blasticidin resistance) in place of Hygromycin resistance. The construct conferring Blasticidin resistance will be introduced to the chromosome using Crispr methodology.
Figure 1. Gene map representation of vector #1217.
In order to examine if the transformation was successful, DNA from eight bacterial colony samples were run using gel electrophoresis. The results are demonstrated below in Figure 2. A band appearing around 1,040 kDa in samples Vector, 3, 5, and 7 is representative of the NheI-NheI fragment conferring Hygromycin resistance. This band indicates that these bacterial samples were not successfully transformed into containing the Blasticidin resistance. In all samples, a band present at 560 kDa is representative of the fragment between HindIII and HindIII. However, an additional band appeared for samples 2, 4, 6, and 8 around 490 kDa. Since it was difficult to distinguish between the band at 560 kDa and 490 kDa, we ran another gel with samples Vectors 2, 3, 4, 6, and 8. The results are demonstrated in Figure 3, where a band at 560 kDa is visible in all samples. Furthermore, there is a clear appearance of a band at 490 kDa in samples 2, 4, 6, and 8. Since the original NheI-NheI Hygromycin resistance sequence was 1,040 kDa long and was replaced with an NheI-NheI fragment for Blasticidin resistance that was 420 kDa long, the final sequence between ClaI and HindIII should appear around 490 kDa. The presence of this band identifies the samples which have been successfully transfected with Blasticidin resistance.
Figure 2. Transformation results examined using gel electrophoresis to compare vector #1217 and eight bacterial samples.
Figure 3. Transformation results repeated using gel electrophoresis. Bacterial samples 2, 4, 6, and 8 demonstrate successful transformation with Blasticidin resistance and, therefore, the degron.
The transformation described above is planned to be used in several mammalian cell types including HeLa, C12, U2OS, and SH-SY5Y cells. To study the effects of the transformation on Codanin-1, Auxin was first introduced into transformed HeLa cells. Interestingly, the knockout of Codanin-1 by the degron did not result in cell death or changes in the cell phenotype. This led to the theory that Codanin-1 is not required in all cell types. Thus, further studies in this area point to the knockout of Codanin-1 in cells in which Codanin-1 is believed to be of significance, such as U2OS or SHSY cells. The effects of Codanin-1 elimination on cellular function following introduction of Auxin in successfully transformed U2OS or SHSY cells has yet to be confirmed.
Previous studies we have conducted have worked towards determining the correct concentration of Hygromycin to use in order to achieve resistance. However, these studies have been unsuccessful as the concentration used have been found to be lethal. Further study of the optimal concentration of Hygromycin should be performed.
SUMMARY:
This research focuses on understanding the properties and interactions of Codanin-1, a protein associated with Congenital Dyserythropoietic Anemia Type I (CDA I). The study investigates the influence of Codanin-1 on the activity and post-translational modification of Asf1, a histone chaperone protein, and explores methods for introducing a degron into the Codanin-1 gene to control protein degradation.
ABSTRACT:
Congenital Dyserythropoietic Anemia Type I (CDA 1); Exploring the Properties and Interactions of Codanin-1
Abigail Razi advised under Dr. Benny Motro and PhD student Moriya Kahta
Congenital Dyserythropoietic Anemia Type I (CDA I) is an autosomal recessive disease associated with abnormalities in erythrocyte precursor proliferation that may be expressed as spongy heterochromatin. The disorder is caused by a mutation in the CDANI gene encoding for the Codanin-1 protein. Codanin-1 is a conserved protein which serves as a protein scaffold and is presumably involved in chromatin assembly and maintenance.
Our research focuses on understanding the function of the Codanin-1 protein. Previously, it was demonstrated that Codanin-1 acts as a protein scaffold to recruit the histone chaperon protein Asf1. Overexpression of Codanin-1 results in a shift in the localization of Asf1 from the nuclear region to the cytoplasm.
In a subsequent analysis of the interactions between Codanin-1 and Asf1, the possible influence of Codanin-1 on post translational modification (PTM) of Asf1 was studied. To do so, HeLa cells underwent an inducible knockout of the gene that codes for Codanin-1. The prediction was that the knockout of this gene would prevent the expression of Codanin-1, thereby influencing the activity and PTM of Asf1. Then, immunoprecipitation was conducted to precipitate Asf1 from treated and untreated cells. Mass spectroscopy was used to identify PTM in Asf1 from cells containing endogenous Codanin-1 and cells containing the inducible knockout of Codanin-1. Differences in PTM between the endogenous Asf1 protein and the Asf1 protein following the inducible knockout of Codanin-1 would not only allude to the influence of Codanin-1 on the activity of Asf1, but more importantly, to larger revelations regarding the role of Codanin-1 in cellular function. The results of this study regarding Asf1 modifications have yet to be identified by mass spectroscopy.
In an additional project, I aimed to change the endogenous Codanin-1 gene by introducing a construct that contained a degron, just prior to its stop codon. A degron is a portion of a protein that is important for controlling protein degradation at the proteasome. In this study, the degron is Auxin (IAA)-dependent, namely in the presence of IAA receptors in the cells. When IAA is added, it binds to its receptor. The receptor recognizes the degron and sends Codanin-1 to the proteasome for degradation.
To insert the plasmid into the genome, a construct was created (referred to as #1217) by adding a 5’ arm and 3’ arm to allow for homologous recombination to occur. Crossover then occurred between the endogenous chromosome and the construct containing the degron and Hygromycin antibiotic resistance. The resulting chromosome contained the same DNA as the endogenous Codanin-1 gene, but also contained the additional degron and antibiotic resistance.
Because there are two chromosomes, it is necessary to introduce the degron into both strands using two constructs that confers cells resistant to both antibiotics: Hygromycin and Blasticidin. The introduction of this gene into the chromosome induces antibiotic resistance by producing a protein that degrades its respective antibiotic, thereby deeming the cell resistant. Since cells will only survive if antibiotic resistance has been introduced, cell survival would indicate successful transformation. In cells where the degron was inserted to both alleles, the addition of Auxin should lead to the degradation of Codanin-1.
Using Crispr methodology, we utilized an sgRNA to cut a strand of chromosomal DNA in order to insert the degron which contained Hygromycin resistance. To insert the degron into the second chromosome, we looked to excise the Hygromycin antibiotic resistance fragment of the plasmid between NheI and NheI and replace it with Blasticidin antibiotic resistance. In order to insert the Blasticidin antibiotic resistant, we edited vector #1217, represented in Figure 1, by inserting a fragment from plasmid #1158 (Blasticidin resistance) in place of Hygromycin resistance. The construct conferring Blasticidin resistance will be introduced to the chromosome using Crispr methodology.
Figure 1. Gene map representation of vector #1217.
In order to examine if the transformation was successful, DNA from eight bacterial colony samples were run using gel electrophoresis. The results are demonstrated below in Figure 2. A band appearing around 1,040 kDa in samples Vector, 3, 5, and 7 is representative of the NheI-NheI fragment conferring Hygromycin resistance. This band indicates that these bacterial samples were not successfully transformed into containing the Blasticidin resistance. In all samples, a band present at 560 kDa is representative of the fragment between HindIII and HindIII. However, an additional band appeared for samples 2, 4, 6, and 8 around 490 kDa. Since it was difficult to distinguish between the band at 560 kDa and 490 kDa, we ran another gel with samples Vectors 2, 3, 4, 6, and 8. The results are demonstrated in Figure 3, where a band at 560 kDa is visible in all samples. Furthermore, there is a clear appearance of a band at 490 kDa in samples 2, 4, 6, and 8. Since the original NheI-NheI Hygromycin resistance sequence was 1,040 kDa long and was replaced with an NheI-NheI fragment for Blasticidin resistance that was 420 kDa long, the final sequence between ClaI and HindIII should appear around 490 kDa. The presence of this band identifies the samples which have been successfully transfected with Blasticidin resistance.
Figure 2. Transformation results examined using gel electrophoresis to compare vector #1217 and eight bacterial samples.
Figure 3. Transformation results repeated using gel electrophoresis. Bacterial samples 2, 4, 6, and 8 demonstrate successful transformation with Blasticidin resistance and, therefore, the degron.
The transformation described above is planned to be used in several mammalian cell types including HeLa, C12, U2OS, and SH-SY5Y cells. To study the effects of the transformation on Codanin-1, Auxin was first introduced into transformed HeLa cells. Interestingly, the knockout of Codanin-1 by the degron did not result in cell death or changes in the cell phenotype. This led to the theory that Codanin-1 is not required in all cell types. Thus, further studies in this area point to the knockout of Codanin-1 in cells in which Codanin-1 is believed to be of significance, such as U2OS or SHSY cells. The effects of Codanin-1 elimination on cellular function following introduction of Auxin in successfully transformed U2OS or SHSY cells has yet to be confirmed.
Previous studies we have conducted have worked towards determining the correct concentration of Hygromycin to use in order to achieve resistance. However, these studies have been unsuccessful as the concentration used have been found to be lethal. Further study of the optimal concentration of Hygromycin should be performed.
SUMMARY:
This research focuses on understanding the properties and interactions of Codanin-1, a protein associated with Congenital Dyserythropoietic Anemia Type I (CDA I). The study investigates the influence of Codanin-1 on the activity and post-translational modification of Asf1, a histone chaperone protein, and explores methods for introducing a degron into the Codanin-1 gene to control protein degradation.
ABSTRACT:
Congenital Dyserythropoietic Anemia Type I (CDA 1); Exploring the Properties and Interactions of Codanin-1
Abigail Razi advised under Dr. Benny Motro and PhD student Moriya Kahta
Congenital Dyserythropoietic Anemia Type I (CDA I) is an autosomal recessive disease associated with abnormalities in erythrocyte precursor proliferation that may be expressed as spongy heterochromatin. The disorder is caused by a mutation in the CDANI gene encoding for the Codanin-1 protein. Codanin-1 is a conserved protein which serves as a protein scaffold and is presumably involved in chromatin assembly and maintenance.
Our research focuses on understanding the function of the Codanin-1 protein. Previously, it was demonstrated that Codanin-1 acts as a protein scaffold to recruit the histone chaperon protein Asf1. Overexpression of Codanin-1 results in a shift in the localization of Asf1 from the nuclear region to the cytoplasm.
In a subsequent analysis of the interactions between Codanin-1 and Asf1, the possible influence of Codanin-1 on post translational modification (PTM) of Asf1 was studied. To do so, HeLa cells underwent an inducible knockout of the gene that codes for Codanin-1. The prediction was that the knockout of this gene would prevent the expression of Codanin-1, thereby influencing the activity and PTM of Asf1. Then, immunoprecipitation was conducted to precipitate Asf1 from treated and untreated cells. Mass spectroscopy was used to identify PTM in Asf1 from cells containing endogenous Codanin-1 and cells containing the inducible knockout of Codanin-1. Differences in PTM between the endogenous Asf1 protein and the Asf1 protein following the inducible knockout of Codanin-1 would not only allude to the influence of Codanin-1 on the activity of Asf1, but more importantly, to larger revelations regarding the role of Codanin-1 in cellular function. The results of this study regarding Asf1 modifications have yet to be identified by mass spectroscopy.
In an additional project, I aimed to change the endogenous Codanin-1 gene by introducing a construct that contained a degron, just prior to its stop codon. A degron is a portion of a protein that is important for controlling protein degradation at the proteasome. In this study, the degron is Auxin (IAA)-dependent, namely in the presence of IAA receptors in the cells. When IAA is added, it binds to its receptor. The receptor recognizes the degron and sends Codanin-1 to the proteasome for degradation.
To insert the plasmid into the genome, a construct was created (referred to as #1217) by adding a 5’ arm and 3’ arm to allow for homologous recombination to occur. Crossover then occurred between the endogenous chromosome and the construct containing the degron and Hygromycin antibiotic resistance. The resulting chromosome contained the same DNA as the endogenous Codanin-1 gene, but also contained the additional degron and antibiotic resistance.
Because there are two chromosomes, it is necessary to introduce the degron into both strands using two constructs that confers cells resistant to both antibiotics: Hygromycin and Blasticidin. The introduction of this gene into the chromosome induces antibiotic resistance by producing a protein that degrades its respective antibiotic, thereby deeming the cell resistant. Since cells will only survive if antibiotic resistance has been introduced, cell survival would indicate successful transformation. In cells where the degron was inserted to both alleles, the addition of Auxin should lead to the degradation of Codanin-1.
Using Crispr methodology, we utilized an sgRNA to cut a strand of chromosomal DNA in order to insert the degron which contained Hygromycin resistance. To insert the degron into the second chromosome, we looked to excise the Hygromycin antibiotic resistance fragment of the plasmid between NheI and NheI and replace it with Blasticidin antibiotic resistance. In order to insert the Blasticidin antibiotic resistant, we edited vector #1217, represented in Figure 1, by inserting a fragment from plasmid #1158 (Blasticidin resistance) in place of Hygromycin resistance. The construct conferring Blasticidin resistance will be introduced to the chromosome using Crispr methodology.
Figure 1. Gene map representation of vector #1217.
In order to examine if the transformation was successful, DNA from eight bacterial colony samples were run using gel electrophoresis. The results are demonstrated below in Figure 2. A band appearing around 1,040 kDa in samples Vector, 3, 5, and 7 is representative of the NheI-NheI fragment conferring Hygromycin resistance. This band indicates that these bacterial samples were not successfully transformed into containing the Blasticidin resistance. In all samples, a band present at 560 kDa is representative of the fragment between HindIII and HindIII. However, an additional band appeared for samples 2, 4, 6, and 8 around 490 kDa. Since it was difficult to distinguish between the band at 560 kDa and 490 kDa, we ran another gel with samples Vectors 2, 3, 4, 6, and 8. The results are demonstrated in Figure 3, where a band at 560 kDa is visible in all samples. Furthermore, there is a clear appearance of a band at 490 kDa in samples 2, 4, 6, and 8. Since the original NheI-NheI Hygromycin resistance sequence was 1,040 kDa long and was replaced with an NheI-NheI fragment for Blasticidin resistance that was 420 kDa long, the final sequence between ClaI and HindIII should appear around 490 kDa. The presence of this band identifies the samples which have been successfully transfected with Blasticidin resistance.
Figure 2. Transformation results examined using gel electrophoresis to compare vector #1217 and eight bacterial samples.
Figure 3. Transformation results repeated using gel electrophoresis. Bacterial samples 2, 4, 6, and 8 demonstrate successful transformation with Blasticidin resistance and, therefore, the degron.
The transformation described above is planned to be used in several mammalian cell types including HeLa, C12, U2OS, and SH-SY5Y cells. To study the effects of the transformation on Codanin-1, Auxin was first introduced into transformed HeLa cells. Interestingly, the knockout of Codanin-1 by the degron did not result in cell death or changes in the cell phenotype. This led to the theory that Codanin-1 is not required in all cell types. Thus, further studies in this area point to the knockout of Codanin-1 in cells in which Codanin-1 is believed to be of significance, such as U2OS or SHSY cells. The effects of Codanin-1 elimination on cellular function following introduction of Auxin in successfully transformed U2OS or SHSY cells has yet to be confirmed.
Previous studies we have conducted have worked towards determining the correct concentration of Hygromycin to use in order to achieve resistance. However, these studies have been unsuccessful as the concentration used have been found to be lethal. Further study of the optimal concentration of Hygromycin should be performed.