1. Role of the p21 Protein in the Fanconi Anemia (FA) Pathway
Mae Shen
Faculty Sponsor: Dr. Niall Howlett

2. Fanconi Anemia - Clinical Description
Congenital malformations
Short stature
Microcephaly (small head)
Micropthalmia (small eyes)
Hypo/hyperpigmentation
Abnormal thumbs
Hematological abnormalities
Bone marrow failure
Acute myeloid leukemia (x800)
- Fanconi Anemia, or FA for short is a rare genetic disorder primarily thought of as a blood disorder.
Patients often have congenital malformations including…(list)
They are affected by hematological abnormalities such as…(list)
Bone marrow failure – leading cause of death among FA patients
By 40 years of age, bone marrow failure occurs 90% of patients
As you can see, FA patients suffer from hematological abnormalities at a much higher rate than the rest of the population that do not have FA.
They are also at a much higher risk for non-hematological abnormalities
Non-Hematological abnormalities
Head and neck squamous cell carcinomas (x2000)
Gynecologic squamous cell carcinomas (x4000)
Benign and malignant liver, brain and renal tumors

3. Fanconi Anemia - Inheritance
Recessive genetic disorder
Biallelic mutations in any one of thirteen genes results in FA
Autosomal and X-linked
Incidence: 1 in 200,000 – 400,000 live births
Higher incidence rate in certain populations such as Spanish Gypsies and Ashkenazi Jews.
FA is an inherited disorder
recessive genetic disorder
Bialleilic mutations in any one of thirteen genes results in FA
So an individual has to receive two copies of a defective FA gene: one from mother, one from father
If both parents are carriers for a mutation in the same FA gene, 25% chance a child will have FA.
It is both autosomal and X-linked
So one of the FA genes is found on the X chromosome
It is a rare disease, with an incidence rate of abouth 1 in 200,000 – 400,000 live births
Athough the incidence rate is higher in certain populations such as Spanish Gypsies and Ashkenazi Jews

4. Why Study Fanconi Anemia?
Link to Cancer
FA is strongly linked to cancer in the general (non-FA) population.
Four FA genes are breast cancer susceptibility genes in the heterozygous state, for example FANCD1/BRCA2.
Target for Cancer Chemotherapy
The FA pathway is a major cellular determinant of resistance to DNA crosslinks.
Oxaliplatin and cisplatin are DNA-crosslinking agents commonly used in cancer chemotherapy.
Drugs that inactivate the pathway could be used in adjuvant cancer chemotherapy.
So why study FA if it is such a rare disease?
FA is strongly linked to cancer in the general (non-FA) population
For example, four FA genes are actually also breast cancer susceptibility genes
So even though in individual need two mutated copies of an FA gene to get FA, having just one mutated copy of one of these four FA genes will result in an increased risk for breast cancer
So the study of FA is really relevant to a broad range of people
FA pathway is an attractive target in cancer chemotherapy
The FA pathway is a major cellular determinant of DNA crosslink resistance
Thus, cells that have a defective FA pathway, such as those of FA patients, are more susceptible to the damage caused by DNA-crosslinking agents.
These abnormal cross-shaped chromosomes are thought to be a result of failure to repair a DNA-crosslink, and are harmful because they prevent proper DNA replication

5. The Fanconi Anemia PathwayUb C G 24 M A F E
100 B
BLM
UBE2T L
ATR
ATRIP
ATM P ? 
PCNA Ub  D2 I Ub D2 I
J/BRIP1
D1/BRCA2
N/PALB2 51
NBS1
BRCA1 Ub
USP1
UAF1
The Fanconi anemia pathway plays a role in repairing damaged DNA.
The D2 Fanconi anemia protein is central to the pathway.
After DNA damage has occurred, what is known as the FA core complex adds a single molecule of ubiquitin to the D2 protein, a key step in the activation of the pathway.
D2 then associates with other downstream proteins to promote the repair of damaged DNA.
Interactions with other proteins also occur but we are primarily concerned with the the regulation of this step.
Despite this, much is still unknown about the pathway and how the monoubiquitination of D2 is regulated.
So these different forms of D2 (D2 itself and monoubiquitinated D2), can easily be detected by a method called western blotting.

6. p21 is Required for DNA damage-inducible FANCD2 monoubiquitination
HCT116
p21+/+
p21 -/-
Hours post UV (20 J/m2)
0.1
0.3
0.6 1 2 6 24
0.1
0.3
0.6 1 2 6 24
FANCD2-Ub
FANCD2
p21 * *
You can detect the different forms of the FANCD2 protein: the non-Ub and mono-Ub by the Western blot method
Western blot:
Method of analyzing protein levels in cells
Grow your cells
Lyse them open
Purify the proteins
Separate the proteins by size by running in an electrophoretic gel
Smaller proteins move faster so they will appear further down than larger proteins
Transfer the proteins to a membrane
Incubate membrane with antibodies that recognize specific proteins
So you can see in this blot that there are clearly two bands: the non-ub on the bottom, the mono-ub on the top
So normally cells contain the p21 protein like these cells, which are HCT116 p21+/+ cells
So you can see here that p21 is present (point to band)
However, in p21-/- cells, p21 is absent, so no band is seen here
And clearly, in these cells that lack p21, mono-ub of D2 is impaired

7. What is p21? Major regulator of the cell cycle Arrests cell cycle
1) Inhibit CDKs
2) Interact with PCNA to halt DNA replication
So what is p21?
Most cells go through cycles of replication and division, and this process is called the cell cycle
The cycle consists of several phases:
G1 – growth phase
S – DNA replication
G2 – another growth phase
M – cell division
P21 is a major regulator of the cell cycle
When conditions are unfavorable (ie. DNA damage) p21 arrests the cell cycle at late G1, preventing progression inito S
It does this in two ways:
1) inhibit CDKs (central components of the cell cycle control system that are required for transition into S phase)
2) interacts with PCNA (protein involved in DNA replication)
This way damage can be repaired before it is replicated
Another important point regarding p21 is that it is a major target of p53
p53 one of most important genes preventing cancer in humans
Illustrated by fact that p53 is mutated in about 50% of cancers
So there is important link between p21 and cancer development
Major target of the tumor
suppressor transcription
factor p53
Figure 8.4 The Biology of Cancer (© Garland Science 2007)

8. Characterize the interaction between
My Project
Goal:
Characterize the interaction between
FANCD2/FANCD2-Ub and p21
Specifically:
Are FANCD2 monoubiquitination and p21 coordinately regulated following DNA damage?
Is p21 required for resistance to DNA crosslinks?
Does inhibition of p21 degradation affect FANCD2 monoubiquitination?
Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination?

9. 1) Are D2 mono-Ub and p21 Coordinately Regulated following DNA Damage?
Rationale:
If the kinetics of D2 mono-Ub and p21 expression are similar, this would be suggestive that both proteins are involved in the same cellular process.
Methods:
Treat cells with DNA damaging agents.
UV irradiation (TT dimer)
Mitomycin C (DNA crosslink)
Observe levels of monoubiquitinated D2 (D2-Ub) and p21 over time.

10. 1) Are D2 mono-Ub and p21 coordinately regulated following DNA damage?
Hours post-UV (20J/m2 )
p21
FANCD2
FANCD2-Ub
U2OS *
Hours post-MMC (100nM)
p21
FANCD2
FANCD2-Ub *
U2OS
- Human osteosarcoma (bone cancer) cell line expressing wild-type p53
FANCD2 and p21 do not appear to be coordinated following DNA damage

11. 2) Is p21 required for resistance to DNA crosslinks?
Rationale:
FA patient cells are hypersensitive to DNA-crosslinks.
Since cells lacking p21 have impaired monoubiquitination of D2, they should exhibit a similar sensitivity.
Methods:
Treat HCT116 p21+/+ and HCT116 p21-/- cells with the DNA-crosslinking agent mitomycin C (MMC).
Allow cells to grow for about 10 days.
Stain with crystal violet to quantify cells that survived.
Observe whether cells lacking p21 (HCT116 p21-/-) are more sensitive to MMC than wild type cells (HCT116 p21+/+).

12. 2) Is p21 required for resistance to DNA crosslinks?
Our results did not show that p21 is required for MMC resistance
p21 does not appear to be required for resistance to MMC

13. 3) Does inhibition of p21 degradation affect FANCD2 monoubiquitination?
Rationale:
p21 is degraded by the proteasome following UV irradiation.
If p21 degradation is inhibited, how is D2 mono-Ub affected?
Methods:
Treat cells with the proteasome inhibitor MG-132 prior to UV irradiation to inhibit p21 degradation.
Observe levels of FANCD2 monoubiquitination.
Background MG-132 is a potent, cell permeable, and selective proteasome inhibitor (Ki = 4 nM), which inhibits NFkappaB activation by preventing IkappaB degradation (IC50 = 3 ¿M). In addition, it blocks degradation of shortlived proteins, which in turn induces HSP and ER chaperone expression, leading to thermotolerance.

14. 3) Does inhibition of p21 degradation affect FANCD2 monoubiquitination?
Hours post-UV (20J/m2 )
No UV
Control
MG-132 (10μM)
p21
FANCD2
FANCD2-Ub
BJ-Tert *
p21
FANCD2
FANCD2-Ub
Hela
p21
FANCD2
FANCD2-Ub
U2OS *
BJ-TERT  immortalized human fibroblasts
The cell line was derived from cervical cancer cells taken from Henrietta Lacks, who died from her cancer on October 4, 1951
FANCD2 monoubiquitination decreases while p21 increases
consistently after treatment with MG-132.

15. 4) Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination?
Rationale:
One of the functions of p21 is to inhibit CDK.
Is this function required for FANCD2 mono-Ub?
Methods:
Treat HCT116 p21-/- cells with roscovitine (a CDK inhibitor) prior to UV irradiation.
Observe whether roscovitine can substitute CDK-inhibiting role of p21 and restore D2 monoubiquitination following DNA damage.

16. Roscovitine does not restore UV-inducible FANCD2 monoubiquitination
4) Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination?
Hours post-UV (20J/m2)
FANCD2
FANCD2-Ub
Control
Roscovitine (5 μM)
HCT116 p21+/+
HCT116 p21- / -
Roscovitine does not restore UV-inducible FANCD2 monoubiquitination

17. Conclusions p21 is clearly important to the FA pathway.
However the interaction between FANCD2 and p21 is complex.
The mechanism by which p21 regulates the monoubiquitination of FANCD2 remains to be uncovered.

18. Thank You! RI-INBRE Leukemia Research Foundation
Fanconi Anemia Research Fund
Dr. Howlett
Julie
Jeanne
Meg
Fred
Bill
Sarah