1. SETD2 and Clear Cell Renal Cell Carcinoma
Abby Kepley

2. SETD2 is a Histone Methyltransferase of H3K36me3
A histone methyltransferase is an enzyme that catalyzes the transfer of 1-3 methyl groups to residues of arginine and lysine within histone proteins. SETD2 is a histone methyltransferase that specifically trimethylates lysine residues on lysine-36 of histone H3. Although it is generally inferred that acetylation and methylation of histones respectively leads to active and inactive DNA, this is not the case for SETD2-mediated methylation. Studies have shown that methylation on lysine and arginine residues on histones 3 and 4 are dependent upon the situation. In the case of SETD2, tri-methylation leads to active chromatin.
H3K36me3
H3 = histone 3
K36 = lysine 36
me3 = tri-methylation

3. Post-translational modifications of H3K36 are important in regulating DNA structure and expression throughout the genome
Given the fact that histone 3 is found within nucleosomes throughout the genome, we can easily form the hypothesis that post-translational modifications of this protein are going to have a profound effect on DNA structure and expression. This is the basic idea of the histone code hypothesis and epigenetics.
It has been noted that SET2, the yeast homolog of SETD2, is responsible for the prevention of aberrant transcriptional initiation, exon definition, the regulation of replication origin firing, DNA replication checkpoint control, and the maintenance of genomic stability through DSB response.
Now we must ask how exactly SETD2 is able to act so strongly in controlling the transcription of genes. This picture shows a scenario in which SETD2 associates with the CTD (C-terminal domain) of RNA Polymerase II as it proceeds through the elongation portion of transcription. As RNA Polymerase moves along, SETD2 carries out its enzymatic activity by tri-methylating H3K36.

4. Multiple domains within SETD2 contribute to its enzymatic function
SETD2 carries out its enzymatic function with the help of four protein domains.
The SET domain is responsible for histone methyltransferase activity, and therefore methylates lysine 36 on histone 3.
It does this with the help of a molecule called S-adenosylmethionine, or SAM. SAM contains a chemically reactive methyl group within its structure, which is transferred to the lysine residue once the two substrates have entered the active site of SETD2.
The other three protein domains assist the SET domain in it’s catalytic function
The Post SET Domain plays a crucial role in substrate recognition and enzymatic activity. Specifically, it brings in C-terminal residues that participate in SAM binding and histone tail interactions.
The function AWS domain is currently unknown, but it is likely that it functions similarly to the Post SET domain
The WW Domain interacts with motifs containing proline, in order to mediate protein-protein interactions. Although it has not been specified for SETD2, it very likely contributes to interaction with the chromatin or RNA pol II.
S-adenosylmethionine (SAM)

5. SETD2/HYPB is required for murine embryonic vascular remodeling
Now that we know what SETD2 does at a molecular and cellular level, we now must ask about its effects on a larger scale. This particular study knocked out SETD2/HYPB in order to determine the physiological function and importance of the gene during development. Signs of developmental defects began at E8.5, with growth retardations. There were no KO embryos viable after E10.5, indicating that disruption of SETD2/HYBP is lethal. Heterozygous mice were viable, and the study gave no indication of developmental defects.
These pictures display results from the study.
Left Side
These pictures demonstrate the use of immunohistochemistry and PECAM-1 antibody, which mark endothelial cells, on WT and KO embryos. WT embryos have a well-structured organization of vessels, whereas the KO embryos have a poorly-developed and abnormal vasculature. The study concluded that the KO mice have defects specifically in the process of angiogenesis, rather than vasculogenesis.
Right Side Picture
This picture shows the results of an in vitro tube formation assay, which was used to determine if SETD2/HYBP siRNA-treated cells could form capillary-like tubules. This experiment was used because it was comparable to the morphogenesis that occurs during angiogenesis. siRNA was used to disrupt the SETD2/HYPB mRNA and prevent translation of the protein. The scramble siRNA functioned as a negative control, and therefore showed normal capillary tube formation over the 17 hour period. HYPB siRNA showed abrogated tubule development.
What are the important conclusions that we can draw from this study?
There is no redundancy
In order to express a mutant phenotype, SETD2 must be completely knocked out.
Whole-mount IHC with PECAM-1 antibody
In vitro tube formation assay with siRNA

6. Clear Cell Renal Cell Carcinoma is the most common form of RCC
~75% of renal cell carcinomas are ccRCC
There are both hereditary and sporadic forms
1/3 of patients present with locally advanced or metastatic disease
What does all of this have to do with cancer?
SETD2 is linked to ccRCC, which is the most common type of renal cell cancer. It arises within the renal parenchyma and is distinguished histologically by cells with a clear cytoplasm.
There are both hereditary and sporadic forms of RCC
Hereditary = VHL disease
1/3 of patients present with locally advanced or metastatic disease
50% of patients will die as a consequence

7. Tumor suppressors VHL, BAP1, SETD2, and PBRM1 are commonly mutated in ccRCC
There are four mutations that are very commonly seen in ccRCC, and they are associated with LOH of tumor suppressors VHL, BAP1, SETD2, and PBRM1.
As you can see from the picture, all of the tumor suppressors lie within the same 3p locus.
I has been noted that in ~90% of ccRCC tumors this 3p locus has been lost.

8. Loss of SETD2 function leads to impaired signaling in the DNA damage response
What does loss of SETD2 function specifically do in ccRCC?
Previously, I mentioned that one of major roles of tri-methylation of H3K36 was “maintenance of genomic stability thorough repair of DSBs.” This study shows that loss of SETD2 function results in impaired signaling in the DNA damage response.
This study used the DNA damage inducing agent etoposide, to cause double strand breaks in SETD2 wt and SETD2 mutant ccRCC cells.
ATM/yH2AX
This picture shows western blots of SETD2 wt and mutant ccRCC cells. Antibodies for each of the proteins indicated on the right were used in the analysis. The results show a lack of yH2AX, pATM, and H3K36me3. yH2AX and ATM both play a major role in responding to DSBs.
yH2AX is histone protein 2A, which is phosphorylated in response to DSBs by ATM. It is thought that phosphorylating of yH2AX makes the chromatin less dense, in order to allow for interaction with the DNA repair machinery.
ATM is a type of kinase that phosphorylates H2AX and interacts with other proteins like the NBS1 subunit to allow for the recruitment of other proteins like p53 to repair the DSB and halt cell cycle progression.
Upon DNA damage ATM will auto phosphorylate and become active; there is always ATM present within the cell

9. It also leads to inactivation of p53
This western blot shows SETD2 wt and mutant ccRCC cell lines. Antibodies for each of the proteins indicated on the right were used in the analysis. The results show a reduction in phosphorylated/active p53 in mutant cells treated with etoposide. There is also reduced expression of tumor suppressor p21, which is downstream in the p53 pathway.
p53 becomes activated in response to DNA damage through phosphorylation. One of its functions is to activate genes encoding p21 to further promote the cell’s response to the DSB.
SETD2 loss of function results in inactivation of proteins that are vital in stopping cell cycle progression in response to DNA damage

10. The role of SETD2 in homologous repair
When tri-methylation of H3K36 has occurred, LEDGF is recruited, and the cell can induce cell cycle arrest and undergo homologous recombination when DSBs occur. Specifically, LEDGF will recruit CtIP, which will repress transcription within the damaged section of DNA and allow resection to occur. After this, RPA, a ssDNA-binding protein will be recruited to allow for the binding of RAD51 so that strand invasion from a homologous chromatid can occur.
LEDGF: A transcription co-activator
CtIP: A transcriptional co-respressor
RAD51: promotes strand invasion of a homologous chromatid
RPA: ssDNA-binding protein; keeps the strand from binding to itself so that RAD51 and other proteins can bind to repair the break
Resection generates a 3’ overhang
Without SETD2 tri-methylation this cannot occur, and the cell undergoes micro-homology mediated end joining, which is prone to error and leads to genome instability.

11. Treatment options for ccRCC
There has yet to be a targeted therapy for histone methyltransferases to reach clinical trials.
Current Therapies Include:
Local: nephrectomy, EBRT
Cytokine Therapy: interferon-alpha, IL-2
Targeted: Sunitinib, Sorafenib
Local:
-Nephrectomy = surgical removal of a kidney
-EBRT = external beam radiation therapy; a form of radiotherapy
Cytokine Therapy/Immunotherapy
-Interferon-alpha and IL-2 generate an immune response against the cancer cells
Targeted:
-Sunitinib = multi-targeted RTK inhibitor
-Sorafenib = tyrosine kinase inhibitor and Raf kinase inhibitor

12. Sources Other Information
Pfister, S. X. (2014). SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability. Cell Reports, 7(6), 2006–2018. doi: Hu, M. (2010). Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling. PNAS, 107(7), 2956–2961. Larkin, J. (2012). Epigenetic regulation in RCC: opportunities for therapeutic intervention? Nature Reviews, 9, 147–155. doi: doi: /nrurol Hakimi, A. (2013). Clinical and Pathologic Impact of Select Chromatin-modulating Tumor Suppressors in Clear Cell Renal Cell Carcinoma. European Urology, 63(5), 848–854. doi: Carvalho, S. (2014). SETD2 is required for DNA double-strand break repair and activation of the p53- mediated checkpoint. eLIFE, 1–19. doi: /eLife National Cancer Institute. (2015). Renal Cell Cancer Treatment (PDQ®). Retrieved from Ramnani, D. (2015). Clear Cell Renal Cell Carcinoma. Retrieved from SMART. (2014). PostSET. Retrieved from SMART. (2014). SET. Retrieved from UniProt Consortium. (2015). Q9BYW2 - SETD2_HUMAN. Retrieved from Fernandes de Almeida, S. (2011). Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36. Nature Structural & Molecular Biology, 18, 977–983. doi: /nsmb.2123 Wagner, E. (2014). Understanding the language of Lys36 methylation at histone H3. Nat Rev Mol Cell Biol . , 13(2), 1–26. doi: /nrm3274
Other Information
RCC as a whole is the 7th most common cancer in women, and 5th in men
SETD2 mutation is seen in ~4% of cases
Most are frameshift and associated with advanced stages of cancer
Metastatic rate for SETD2 mutant tumors is 36%
There are >200,000 cases per year
Symptoms/Signs
Early cancer does not typically show any
Hematuria
Low back pain
Mass on the lower back
Anemia
Fever
Fatigue
Weight loss