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 Genomics

 This unit focuses on the use of genomic and proteomic tools to clone and identify the function of the tumor suppressor BRCA1.  We will rely heavily on primary literature to introduce these techniques and explore both their strengths and limitations.  This unit also uses bioinformatics tools.  There will be three exercises in which you will use these tools to answer the same questions the authors used.

We will spend one day exploring each paper and the theory behind the techniques used.  In each article I have numbered the paragraphs.  Individual questions are often accompanied by the symbol which indicates which paragraph of the article relates to the question.  This also makes it easier for us to find the paragraphs during lecture.

Some of the articles are available on-line and have been linked.  Further information can also be found at Online Mendellian Inheritance in Man (OMIM).

 

BRCA Bibliography

 

Genomics and Proteomics Unit: BRCA

1. Albertsen, H. M., et al.:  A physical map and candidate genes in the BRCA1 region on chromosome 17q12-21. Nature Genet. 7: 472-479, 1994.  

In this article the authors create a 4 cM physical map of chromosome 17, and identify 20 potential genes within this region.

The focus of this lecture will be on how a physical map was created, and what criteria were used to identify potential genes within this map.

    1. What are meiotic breakpoints, and how are they used to make a genetic map? 1

    2. "High density genetic maps were created using genetic, radiation hybrid and FISH data."  How would these markers and genes be used to identify the location of the BRCA gene? 2

    3. What is the major difference between a genetic map and a physical map? 

    4. The authors created a map comprised of a contig of 137 overlapping YAC and P1 clones.  How did they use 112 PCR markers to create the contig?  How would the data in Figure 1 be used to accomplish this task? 3

    5. "Several clones were identified that formed small islands of overlapping DNA fragments.  With the map seeded in this way, the islands were extended and adjacent islands bridged by walking." 3  What is walking, and how was it used to obtain YAC and P1 clones that filled in gaps between existing clones?

    6. How did the authors use FISH (7) and fluorescently labeled P1 clones (Table 1) to verify the order of the contig shown in Figure 2?

    7. How many and which potential genes have the authors narrowed their search to?  How do they know that the BRCA gene must be between D17S78 and D17S776? 8

    8. How did the authors obtain clones of each of these genes for sequence analysis? Were any of these genes related to known genes? 8

    9. Which genes were screened for mutations?  Based upon the data, could these genes be BRCA1? 11

    10. What do the authors conclude from their work?

 

3.  Wu, L. C., et al :  Identification of a RING protein that can interact in vivo with the BRCA1 gene product. Nature Genet.  14: 430-440, 1996. 4023

In this article the authors examine which domains of BRCA1 interact with other proteins.

The focus of this lecture will be on detecting protein-protein interactions by using mutagenesis and the yeast two hybrid system.

    1. What is a RING motif?  What possible functions do the authors assign to this motif? 2

    2. What percent amino acid identity is observed between mouse and human BRCA1?  3  Which two domains are conserved?  What is the proposed function of these domains?

    3. How did the authors find BARD1? 4   Why did they want to find a protein like BARD1?

    4. What did the authors use as bait in their yeast two-hybrid screen?  5  What was this fused to in the plasmid?

    5. What was used as the fish in the yeast two-hybrid screen and what was it fused with?  How many clones were screened and how many reacted with BRCA1?  How many were identified as unique mRNAs? 5

    6. How did the authors determine whether or not the same proteins reacted with BRCA1 in mammalian cells in vivo?  7    Which clone was active?  Fig. 1.

    7. How did the authors confirm that the same proteins reacted with BRCA1 using immunoprecipitation?  How did they manage this without antibodies against BRCA1?  Was the same clone active? 8    Fig. 2.

    8. Upon sequencing BARD1, what regions of homology does it have with BRCA1?  What unique regions does it possess?  What is a BRCT domain?  9 and 10, Figs. 3 and 4.

    9. How did the authors produce BARD1 and BRCA1 in vitro and show that they could interact outside of a cell?  11, Fig. 5.

    10. Where is BARD1 produced in the body and which chromosome is it found on?  13.

    11. How did the authors determine which regions of BARD1 and BRCA1 were interacting with each other?  14 and 15, Figs. 6 and 7.

    12. How did the authors link mutations in BRCA1 that are associated with breast cancer with BRCA1 binding to BARD1?  16 and 17, Fig. 8.

    13. What is the significance of this work? 

     

    BRCA1 Bioinformatics Assignment

 

4.  Hakem, R., et al: The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell 85: 1009-1023, 1996.

In this article the authors examine the role of BRCA1 in vivo by generating knockout (KO) mice.

The focus of this lecture will be on how to use model organisms to study the function of a gene.

 

    1. Why make a BRCA1 KO mouse?  Why not a mouse that over-expresses BRCA1?

    2. How were BRCA1 KO mice generated?  6, Fig. 1

      How were embryonic stem (ES) cells used?

      Which exons were deleted?

      How were KO cells selected? 

      What fraction the ES cells had one copy of BRCA1 deleted?

      How was the deletion confirmed by Southern Blot (explain the difference in sizes of the wt and mt bands)?

      How was the deletion confirmed by PCR (why did primer pair a/b amplify only the wt and primer pair c/d only the mt)?

    3. How were heterozygous BRCA1 mice generated?  7

    4. What is the phenotype of heterozygous BRCA1 mice?  8  Fertile or sterile?  Get cancer?

    5. What is the phenotype of homozygous BRCA1 KO mice?  Are they viable?  If not, when do they die?  9 and 10. 

    6. What happens to BRCA1 expression during development?  13, Fig. 4.

    7. What evidence supports the author's claim that BRCA1 KO do not have an increase in apoptosis?  21

    8. What evidence supports the author's claim that BRCA1 KO do have a decrease in growth?  22, Fig. 6.

    9. At which stage in the cell cycle is growth arrested?  Which proteins are involved?  23 & 24.

    10. How are BRCA1 and mdm-2 KO cells similar in phenotype?  How is the mdm-2 phenotype regulated by mutations in p53?  27

    11. In BRCA1 KO cells, what happens to mdm-2, p53 and p21 expression?  28 and 29. 

 

 

5.  David Cortez, Yi Wang, Jun Qin, Stephen J. Elledge.  Requirement of ATM-Dependent Phosphorylation of Brca1 in the DNA Damage Response to Double-Strand Breaks. Science 268: 1162-166, 1999.

In this article the role of ATM in the phosphorylation and activation of BRCA1 is investigated. 

This paper also introduces a powerful new proteomics tool, MALDI-TOF.

  1. Cells with mutations in BRCA1, BRCA2 and ATM have what phenotype in common? 1

  2. What activates ATM?  What activity does it have?  What other proteins does it activate? 2

  3. BRCA1 mutations in mice affect which cellular processes? 3

  4. ATM was identified as a protein bound to BRCA1 in cells using what technique?  Looking at figure 1A, why would researchers have to use this more sensitive technique? 4

  5. What happens to BRCA1 in cells in response to treatment with g-irradiation? Is this dependent on ATM? Fig. 1B and 1C, 4 and 5.

  6. How did the authors narrow down which serines were being phosphorylated on BRCA1? 6

  7. What is an SQ cluster?  What does it have to do with ATM and BRCA1? 7

  8. How were mass spectroscopy and CIP used to narrow down the region of BRCA1 peptide 1351-1552 that was phosphorylated?  How was MALDI-TOF used to specifically identify the serines that were phosphorylated? 8, Figure 3A-C.

  9. Is this phosphorylation dependent on ATM?  9, Fig. 3D-F.

  10. BRCA1 -/- cells were transformed with wt or A1432A/S1524S BRCA1 and exposed to g-irradiation.  What can be concluded about the phosphorylation of these residues? 10, Fig. 4D.

  11. Diagram a pathway illustrating how BRCA1 is activated when double stranded breaks occur in a cell’s chromosomes.

 

6. Roger A. Greenberg, Bijan Sobhian, Shailja Pathania, Sharon B. Cantor, Yoshihiro Nakatani, and David M. Livingston.  Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes.  GENES & DEVELOPMENT 20:34–46, 2006

This paper examines the “super complexes” formed by BRCA1 in response to DNA damage.

  1. BRCA1 has been shown to be involved in multiple cellular responses required to repair DNA damage including:  2
  1. What is the evidence that BRCA1 interacts with multiple proteins? 3
  1. What is the evidence that the cellular location of BRCA1 changes in response to DNA damage? 4
  1. In addition to ATM, what other proteins phosphorylate BRCA1? 5
  1. How were lasers used to identify proteins that required BRCA1 to localize to regions with double strand breaks?  Which proteins required BRCA1 and which did not? 7-9, Fig. 1
  1. How were proteins in complexes with BRCA1/BARD1 identified?  Which proteins bound to the complex in response to DNA damage? 11-13, Fig. 2
  1. Was phosphorylation of BRCA1 necessary for protein binding?  Fig. 3A looks at ATM -/- cells in the presence and absence of a CHK2 inhibitor.  Which proteins bind to BRCA1 as a result of ATM dependent phosphorylation?  Which bind as a result of CHK2 dependent phosphorylation? 14-15.
  1. Figure 3B looks at CHK2 -/- cells in the presence and absence of an ATM inhibitor.  Is TopBP1 binding dependent on ATM or CHK2 phosphorylation of BRCA1? 15
  1. What evidence is provided that BRCA1 is actually found in several different complexes in the cell? What groups of proteins are in the two complexes? 17-20, Fig. 4A and B.
  1. How does BRCA1 control the G2/M checkpoint?  Is BACH1 or CtIP involved?  21-22 Fig. 5A
  1. There is an origin of replication near the b-globin gene cluster.  How as ChIP used to identify which proteins bound near this origin of replication?  Which proteins did bind?  Does this regulate the G2/M or G1/S checkpoint?  26, Fig. 6A
  1. Based on this data, what roles does BRCA1 play in a cell?  Fig. 7

 

7.  Hedenfalk, I., et al:  Gene-expression profiles in hereditary breast cancer. New Eng. J. Med. 344: 539-548, 2001.

In this article the authors examine differences in gene expression in tumors from carriers of BRCA1 or BRCA2 mutations and sporadic cases of breast cancer.

The focus of this lecture will be the use of microarrays to identify key genes involved in cancer.

    1. The authors found how many genes showed variation in expression out of the 3226 genes screened?  6

    2. Fig. 1B shows grouping of different tumors if the expression of all 3226 genes are accounted for.  Fig. 1C shows the same grouping with just the best genes used.  Was this effective as a test for BRCA1 or BRCA2 mutations?  Which mutations were detected with the most accuracy?  7 and 8.

    3. What major cellular processes are regulated by the genes that are differentially expressed in BRCA1 or BRCA2 mutation-positive tumors?  What is the impact of the expression of these genes on a cell?  9 and 10.

    4. What is the difference between a gene-expression microarray and a tissue microarray?  Were the results from the two microarrays consistent with each other?  11 and 12, Figs 1D & 1E.

    5. One patient with sporadic breast cancer was mis-diagnosed as having a BRCA1 mutation.  What did the authors discover about this patients BRCA1 gene that explained the decrease in BRCA1 gene expression?  13 & 14, Fig. 4

    6. What are three important discoveries from this research?  See Discussion

 

8.  Flemming, MA, et al.; Understanding missense mutations in the BRCA1 gene: An evolutionary approach.  PNAS 100:1151-1156 (2003).

In this article the authors examine the evolution of BRCA1 in 57 mammals. 

The focus of this lecture will be on how we can infer the function of a region of a gene by comparative genomics and molecular evolution.

  1. What fraction of mutations in BRCA1 are frameshifts?  How many missense mutations have been identified? 1
  1. Why is comparative phylogenetic analysis useful in studying BRCA1? 2
  1. Were the DNA or protein sequences from the 57 species aligned?  Why? 4
  1. How were regions evolving under positive selection defined?  Why do you think they used this definition? 10
  1. What percent of the BRCA1 amino acids used in this study were fixed?  Showed conservative substitutions? 14
  1. Why were chicken (Gallus) and frog (Xenopus) sequences used to help identify conserved regions? 25
  1. Regions evolving under positive selection showed changes in primates in locations that were conserved in non-primates.  Where did these changes occur? 27

 

Optional web assignment:

Here are the accession numbers of seven of the species used in this paper.

U14680 OR AF019075 OR AF19076 OR U50709 OR AF284018 OR AF284017 OR AF355273

If you copy and paste this into Biology Workbench/Ndjinn and select GBPRI, GMAM, GBVRT, you should be able to get all 7 sequences in a single search.

Align these sequences and look for regions conserved in all species, in mammals, and in primates.  This is the type of data the researchers analyzed in this paper.

 

 

    Other Genomics Links 

 

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