Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation

Q: What was used for the sequencing library?

The input and ChIP DNAs (5 ng for transcription factor ChIPs, 20 ng for histone modification ChIPs) were used for preparation of sequencing libraries according to the manufacturer’s instructions for the Genome Analyzer GAII instrument (Illumina).

Q: What is the morphological and immunohistochemical characteristics of cal51 cells?

CAL51 cells exhibit morphological and immunohistochemical characteristics of epithelial mammary cells, are tumorigenic in nude mice, and havewild-type p53 and a normal karyotype.

Q: What is the main reason for the mutated p53 in solid tumors?

Evidence suggests that in solid tumors that harbor mutated p53, mitotic catastrophe takes over and becomes the major form of cell death induced by radiotherapy (61, 64).

Q: What is the role of lncRNAs in biological processes?

Evidence suggests that lncRNAs play substantial regulatory roles in diverse biological processes, such as transcriptional regulation, genetic dosage compensation, cell cycle regulation, and development [for a recent review, see (28)].

Q: What is the key regulator of the transcriptional response to IR?

Control of the transcriptional response to IR by p53, NF-kB, and ATM Pathway analysis indicated that the group of IR-induced genes was highly enriched for p53-associated signaling pathways (fig. S2A), suggesting that p53 is the key regulator of the transcriptional response to IR.

Q: What primers were used for known genomic target regions?

Detection primers for known genomic target regions were as follows: MDM2 (a target of TP53), 5′-CGTTCCGAAACTGCAGTAAA-3′ (forward) and 5′-CAGCTGGAGACAAGTCAGGA-3′ (reverse); ICAM1 (a target of RelA), 5′-GCCGCCCGATTGCTTTAG-3′ (forward) and 5′- GCTGCAGTTATTTCCGGACTG-3′ (reverse); and CDKN1A (for H3K4me1 and H3K4me3), 5′-TGCGTTCACAGGTGTTTCTG-3′ (forward) and 5′-CACATCCCGACTCTCGTCAC-3′ (reverse).

Question

1. What are the contributions in "Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation" ?

2. What is the role of aTF3 in NF-kB?

3. What is the key transcription factor that regulates the transcriptional response to IR?

4. How many p53 targets were detected at 4 hours after IR?

Accepted Answer

Althoughmuchof this responseoperates at the protein level, a critical component of the network sustains many DDR branches by modulating the cellular transcriptome.. The authors identifiedprotein-codingandpreviouslyunidentifiednoncodinggenes that were responsive to IR, and demonstrated that IR-induced transcriptional dynamicswasmediated largely by the transcription factors p53 and nuclear factor kB ( NF-kB ) andwas primarily dependent on the kinase ataxia-telangiectasia mutated ( ATM ).. The resultant data set provides a rich resource for understanding a basic, underlying component of a critical cellular stress response.. Using deep sequencing, the authors delineated three layers in the transcriptional response to IR inhumanbreast cancer cells: changes in theexpressionof genesencodingproteinsor longnoncoding RNAs, alterations in genomic binding by key transcription factors, and dynamics of epigenetic markers of activepromoters andenhancers.

Accepted Answer

In macrophages, activating transcription factor 3 (ATF3) was identified as a transcriptional repressor that mediated the rapid decline in expression of NF-kB target genes after their induction by immune stimulation.

Accepted Answer

The other pivotal transcription factor that regulated the transcriptional response to IR in their study is NF-kB, whose activation in response to DSB induction is also ATM-dependent and carried out by an ATM-controlled loop involving several proteins [reviewed in (67)].

Accepted Answer

Of the 299 induced genes at 4 hours after IR, 138 genes (46%) were detected by ChIP-seq as p53 targets (about 10-fold enrichment; P< 10−99, tail of hypergeometric distribution).

Accepted Answer

The input and ChIP DNAs (5 ng for transcription factor ChIPs, 20 ng for histone modification ChIPs) were used for preparation of sequencing libraries according to the manufacturer’s instructions for the Genome Analyzer GAII instrument (Illumina).

Accepted Answer

CAL51 cells exhibit morphological and immunohistochemical characteristics of epithelial mammary cells, are tumorigenic in nude mice, and havewild-type p53 and a normal karyotype.

Accepted Answer

Evidence suggests that in solid tumors that harbor mutated p53, mitotic catastrophe takes over and becomes the major form of cell death induced by radiotherapy (61, 64).

Accepted Answer

Evidence suggests that lncRNAs play substantial regulatory roles in diverse biological processes, such as transcriptional regulation, genetic dosage compensation, cell cycle regulation, and development [for a recent review, see (28)].

Accepted Answer

Control of the transcriptional response to IR by p53, NF-kB, and ATM Pathway analysis indicated that the group of IR-induced genes was highly enriched for p53-associated signaling pathways (fig. S2A), suggesting that p53 is the key regulator of the transcriptional response to IR.

Accepted Answer

Detection primers for known genomic target regions were as follows: MDM2 (a target of TP53), 5′-CGTTCCGAAACTGCAGTAAA-3′ (forward) and 5′-CAGCTGGAGACAAGTCAGGA-3′ (reverse); ICAM1 (a target of RelA), 5′-GCCGCCCGATTGCTTTAG-3′ (forward) and 5′- GCTGCAGTTATTTCCGGACTG-3′ (reverse); and CDKN1A (for H3K4me1 and H3K4me3), 5′-TGCGTTCACAGGTGTTTCTG-3′ (forward) and 5′-CACATCCCGACTCTCGTCAC-3′ (reverse).

Accepted Answer

binding of various cofactors and p53’s rich and dynamic PTM repertoire are other important determinants of the outcome of p53 binding (58–60).

Accepted Answer

This decrease in H3K4me1 probably reflects nucleosome displacement associated with the IR-induced binding of p53 to these enhancer regions.

Accepted Answer

67% (12 of 19 genes) of the cluster of p53-independent IR-induced genes were detected as direct RelA targets (fig. S7D), demonstrating the role of NF-kB in the induction of this response.

Accepted Answer

IRinduced p53 binding was also detected near the promoters of five of the eight IR-induced lncRNAs, flagging them as newly identified p53 direct target genes in the DDR (fig. S4C and table S7).

Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation

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Most frequently asked questions

1. What are the contributions in "Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation" ?

2. What is the role of aTF3 in NF-kB?

3. What is the key transcription factor that regulates the transcriptional response to IR?

4. How many p53 targets were detected at 4 hours after IR?

5. What was used for the sequencing library?

6. What is the morphological and immunohistochemical characteristics of cal51 cells?

7. What is the main reason for the mutated p53 in solid tumors?

8. What is the role of lncRNAs in biological processes?

9. What is the key regulator of the transcriptional response to IR?

10. What primers were used for known genomic target regions?

11. What are the main determinants of the outcome of p53 binding?

12. What is the effect of IR on p53?

13. How many of the IR-induced genes were detected as direct RelA targets?

14. What is the significance of IR-induced p53 binding?

Figures

Citations

The transcription factor p53: Not a repressor, solely an activator

The Nuclear Factor kappaB Pathway: A Link to the Immune System in the Radiation Response

The emerging role of lysine demethylases in DNA damage response: dissecting the recruitment mode of KDM4D/JMJD2D to DNA damage sites.

Gene regulation by the tumor suppressor p53 – The omics era

Dynamics of chromatin accessibility and epigenetic state in response to UV damage.

References

Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.

Ultrafast and memory-efficient alignment of short DNA sequences to the human genome

Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc.

An integrated encyclopedia of DNA elements in the human genome

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