Chromatin reader domains of DNMT-targeting protein, UHRF1, are responsible for cancerous DNA hypermethylation

Tue, 08/20/2019 - 08:58

Expression of DNMT2 in human colon tissue shows cytoplasmic and nuclear staining in glandular cells, IHC

By Jamshed Arslan, Pharm. D., PhD.

DNA methylation represses transcription of many genes, including tumor suppressor genes. A protein called UHRF1 recruits DNA methyltransferases (DNMTs) to establish and maintain DNA methylation. UHRF1 has several domains, most notably:

  • N-terminal ubiquitin-like (UBL) domain
  • TTD (tandem Tudor domain) and PHD (ubiquitin-like, containing plant homeodomain) domains that recognize methylation at histones H3K9 (Histone H3 Lysine 9) and H3R2 (Histone H3 Arginine 2), respectively
  • SRA (SET and RING associated) domain that binds to hemimethylated DNA
  • C-terminal RING domain that requires UBL to confer E3 ubiquitin ligase function.

Explore Epigenetic-Machinery

Depleting UHRF1 with or without DNMT inhibition    reduces DNA methylation, thereby reactivating tumor suppressor genes and suppressing cancers. Studies on re-establishing DNA methylation by using loss-of-function mutant UHRF1 domains are unable to indicate if such DNA re-methylation is purely anew. So, research institutes in USA    and China set out to define the UHRF1 domains that maintain cancerous DNA methylation. Using colorectal cancer cells, the team found that chromatin reader domains of UHRF1 maintain cancerous DNA hypermethylation. They found that both histone- and hemimethylated DNA-reader domains (PHD and SRA, respectively) are crucial for colorectal cancer's aberrant DNA methylation. Disrupting SRA or PHD impairs the cancerous potential of colorectal cancer cells by reversing DNA hypermethylation and reactivating tumor suppressor genes.

Sheep Anti-Human DNMT1 Antigen AffinityDNMT1 was detected in HCT 116 human colorectal carcinoma cell line with Sheep Anti-Human DNMT1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6110) at 10 µg/mL for 3 hours at room temperature. Cells were stained with NorthernLights 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Untreated (left) and treated (right) with 1 μM 5-azacytidine for 24 hours. Specific staining was detected in the nuclei and cytoplasm, and nuclear staining was reduced by 5-azacytidine treatment.

UHRF1 chromatin reader domains contribute to DNA hypermethylation in colorectal cancer cells

To dissect the role of UHRF1, the researchers exogenously expressed UHRF1 transgene in human colorectal cancer cells while simultaneously blocking endogenous UHRF1 through shRNA. Such replacement of endogenous UHRF1, allowed the team to observe exogenous UHRF1’s DNA methylation maintenance capacity. It turned out that UHRF1 cannot maintain genomic and cancer-specific DNA methylation when loss-of-function or deletion mutants of PHD domain, but not TTD domain, are used. Similarly, SRA domain mutants could not recognize hemimethylated DNA. Most notably, only the PHD or SRA mutants produced DNA methylation patterns similar to endogenous UHRF1 depletion. A dramatic cancer-specific DNA demethylation was visible in PHD or SRA mutants. In other words, cancerous DNA methylation and the consequent silencing of tumor suppressor genes are dependent on PHD or SRA domains.

The next step was to explore the effects of either domain on the oncogenic properties of colorectal cancer cells.

UHRF1 histone- and hemimethylated DNA-reader domains contribute to the oncogenic properties of colorectal cancer cells

The researchers transplanted colorectal cancer cells with TTD, PHD, TTD-PHD, SRA or RING mutants into immunocompromised mice. TTD-PHD and SRA mutants markedly reduced tumor burden with enhanced tumor latency relative to wild-type or RING mutants. Interestingly, reduction in tumor burden and colorectal cancer cell proliferation in PHD mutants was similar to TTD-PHD double mutants. In other words, SRA and PHD domains are enough to confer oncogenic properties to UHRF1 in colorectal cancer cells. This was corroborated by increased expression of various colorectal cancer metastasis-related genes like ICAM4, PRSS8, JDP2 and FBLN2 in the mutants.

The translational value of these findings was evident when tissue samples from 120 colorectal cancer patients were found to have elevated UHRF1 RNA expression relative to individuals without colorectal cancer. Among the colorectal cancer patients, those with UHRF1 overexpression had reduced progression-free survival.

In sum, UHRF1 maintains cancer-specific DNA methylation for silencing tumor suppressor genes, primarily through its histone reader (PHD) and hemimethylated DNA reader (SRA) domains. This contributes to the malignant potential of colorectal cancer.

Significance for UHRF1 focused therapies in colorectal cancer

Reducing cancerous DNA methylation is important for optimal therapy. Although DNMT inhibitors and aromatic ring of TTD domain have been the focus of therapeutics, this research highlights the prognostic and therapeutic significance of PHD and SRA domains of UHRF1 in human colorectal cancer.

Novus Antibodies Used in this Study

DNMT3A expression in 293 cell lysate, WB

Western blot: DNMT3A Antibody (64B1446) [NB120-13888] - Analysis of (A) Dnmt3a transfected 293 cell lysate and (B) untransfected 293 cell lysate using Dnmt3a antibody at 1 ug/mL.

Fibulin-2 expression in 293T, A431, HeLa and HepG2 cell lysates, WB

Western Blot: Fibulin 2 Antibody [NBP1-33479] - Various whole cell extracts (30 ug) were separated by 5% SDS-PAGE, and the membrane was blotted with Fibulin 2 antibody diluted at 1:1000.

Jdp2 expression human kidney, IHC

Immunohistochemistry-Paraffin: JDP2 Antibody [NBP2-31775] - Staining of human kidney shows strong nuclear positivity in cells in tubules.

LXN expression in lysate from LXN transfected HEK293T cells, WB

Western Blot: LXN Antibody (1E10) [NBP2-01382] - HEK293T cells were transfected with the pCMV6-ENTRY control (Left lane) or pCMV6-ENTRY LXN (Right lane) cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates (5 ug per lane) were separated by SDS-PAGE and immunoblotted with anti-LXN.

Prostasin-Prss8 expression in paraffin-embedded Ca922 xenograft, IHC

Immunohistochemistry-Paraffin: Prostasin/Prss8 Antibody [NBP1-31592] - Paraffin-embedded Ca922 xenograft. Prostasin antibody [N1C2] d ilution: 1:250.

Jamshed Arslan Jamshed Arslan, Pharm D, PhD   
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.


Kong, Xiangqian, et al. "Defining UHRF1 Domains that Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties." Cancer Cell, vol. 35, no. 4, 2019, pp. 633–648.


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