CHFR (Checkpoint with fork-head associated and ring finger)

2014-12-01   Ayse E Erson-Bensan , Hesna Begum Akman , Elizabeth M Petty 




Growing evidence in mice, primary human tumors, and mammalian cell culture models indicate that CHFR may function as a potent tumor suppressor. CHFR functions as part of an early G2\/M checkpoint, more specifically in antephase. Antephase refers to late G2 when chromosome condensation starts. This early mitotic checkpoint causes a delay in chromosome condensation in response to mitotic stresses. The human CHFR gene was originally identified during a search for novel cell cycle checkpoint proteins that have fork-head associated domains. Initial analysis indicated that the CHFR-associated G2\/M checkpoint was inactivated in a subset of cancers as demonstrated by high mitotic indices (a high percentage of cells that have condensed chromosomes) in response to exposure to the microtubule poison, nocodazole, due to lack of CHFR expression or CHFR mutations in various cancers. Many other studies showed promoter hypermethylation leading to low\/no expression of CHFR.



The CHFR gene spans approximately 47 kb and has at least 18 exons (BC012072 vs. NT_024477) as predicted according to Spidey ( While multiple splice forms have been demonstrated (Toyota et al, 2003), the genomic structure has not been experimentally confirmed.


CHFR mRNA is 3189 bp (BC012072). Transcripts that lack exon 2, exon 5 and exon 6 have been detected in various tissues including bone marrow, small intestine, lung, heart, testis, kidney, stomach and lympocytes as well as some cancer cell lines by RT-PCR. Northern blot transcript analysis suggests that limited if any alternative splicing is present in most fetal and adult tissues where CHFR is expressed a prominent 3.2 kb is observed. CHFR mRNA is detected in heart, brain, placenta, lung, liver, muscle, kidney, pancreas by Northern blot analysis (Scolnick and Halazonetis, 2000).
Alternative mRNA transcripts lacking specific exons (2, 5, and/or 6) have been described for CHFR (Toyota et al. 2003) The isoform that lacks exon 2 happens to lack the FHA domain and was also found to be highly expressed in cancer cells when compared to normal samples .


No known pseudogene has been reported


Atlas Image
Domains of CHFR. The forkhead-associated domain of CHFR is located at the N-terminus. RING-finger domain with the ubiquitination activity is located through 303-346 amino acids. Poly-ADP-ribose binding zinc finger motif overlaps with the cystein-rich region near the C-terminus.


CHFR encodes a 652 amino acid protein (according to BC012072 nucleotide sequence) with FHA (forkhead associated), RING (really interesting new gene) finger and cysteine rich domains. Cysteine rich region further harbors a PBZ domain. No alternative isoforms have been described to date.


  • FHA domains (16-123) are present in cell cycle checkpoint genes, transcription factors, protein kinases and have roles in protein-protein interactions with specificity for phosphorylated targets. The three dimensional structure of CHFR suggests that CHFR may be able to recognize as of yet unidentified phosphorylated targets targets (Stavridi et al., 2002; Tsai 2002)..
  • RING finger domains are found in ubiquitin ligases. Ubiquitin ligases attach ubiquitin to target proteins during a cascade of enzymatic reactions. RING finger domains are present in a variety of proteins (e.g. Anaphase promoting complex, APC, Cbl family proteins, MDM2) implicated in cancer.
  • Cys: Cystein-rich region (476-641)
  • PBZ: poly-ADP-ribose binding zinc finger motif (620-644) is at the C terminus. PBZ domain allows CHFR to bind to poly (ADP-ribose). This domain is generally required for the activity of checkpoint response proteins (Ahel et al., 2008)
  • Expression

    CHFR is ubiquitiously expressed in normal fetal and adult human tissues. Protein levels are predicted to fluctuate during the cell cycle possibly through auto-ubiquitination based on overexpression studies in cancer cell lines (Chaturverdi et al., 2002; Kim et al., 2011). Upon mitotic stresses, CHFR protein levels are thought to be stabilized and reaching the highest levels at G2/M.


    Various lines of evidence suggest different cellular localizations for CHFR. Endogenous and low ectopic expression of CHFR showed cytoplasm and spindle localization patterns during mitosis. Higher expression of ectopic CHFR correlated with a shift in the localization to the nucleus (Burgess et al., 2008). Later on, nuclear presence of CHFR was explained via a short lysine-rich stretch (KKK) at amino acid residues 257-259 (Kwon et al., 2009).
    Egeberg et al., 2012 suggested a centrosome/primary cilium axis localization of CHFR. CHFR was also shown to localize to the mitotic spindle by an interaction with TCTP, a protein involved in microtubule stabilization and ?-tubulin (Kim, 2011)


    Initially, CHFR was described to induce an early G2/M checkpoint in response to mitotic stress (Scolnick and Halozenetis, 2000). Cell lines expressing wild-type CHFR exhibit low mitotic index (percentage of cells with condensed chromosomes) and delayed entry into metaphase when centrosome separation is inhibited by mitotic stress. In contrast, cancer cell lines lacking CHFR function enter metaphase without delay and demonstrate higher mitotic indices compared to the CHFR expressing cell lines. (Erson and Petty, 2004).

    In vitro studies suggest that the RING finger domain in CHFR also facilitates ubiquitin ligase function and that it is essential for checkpoint function of CHFR (Chaturved et al., 2002).. In vitro Xenopus extract experiments suggested that CHFR specifically targets PLK1 (polo-like kinase 1) for degradation when extracts are supplemented with high ubiquitin concentrations (Kang, 2002). Thus, according to this in vitro model, CHFR is able to halt cell cycle progression early in mitosis by degrading PLK1, a major player for the activation of mitosis promoting factor. In addition, AURORA A is known to phosphorylate and activate PLK1 as well as CDC25B eventually driving CYCLIN B/CDK1 activation. Interestingly, CHFR was also found to bind via its cysteine rich C-region and ubiquitinate AURORA A, leading to its degradation (Yu et al., 2005). The auto-ubiquitylation ability of CHFR at G2 Phase was proposed to be required for the accumulation of Plk1 and mitotic entry in mammalian cells (Kim et al., 2011). Earlier, Oh et al., showed deubiquitination of Chfr, by USP7/HAUSP (deubiquitinating enzyme) also to regulate its own stability and activity (Oh et al,. 2007).

    On the contrary, Summers et al. suggested PLK1 and AURORA A levels not to change when CHFR was expressed in HCT116 cells treated with Nocodazole (Summers et al., 2005).

    More recently, other proteins including TOPK and PTEN have been shown to play a role in the CHFR related mitotic spindle checkpoing (Shinde et al. 2013)

    Furthermore, Bothos et al., showed that CHFR was able to activate the p38 stress kinase pathway, which reverses chromosome condensation and induces a mitotic arrest and suggested that the ubiquitin ligase function of CHFR may be different than the current in vitro model and that instead of Lys48 ubiquitination, CHFR may link ubiquitin to target protein or proteins via Ly63 due to its interaction with the heteromeric ubiquitin conjugating enzyme complex, Ubc13-Mms2 (Bothos et al., 2003). In the canonical ubiquitin/proteasome pathway, Lys48 is a signal for degradation of target proteins whereas Lys63 ubiquitination functions as a non-proteolytic tag for protein targets. Lys63 ubiquitination is thought to be involved in DNA repair mechanisms. Indeed, CHFR appears to have important roles in DNA damage response (Shtivelman et al., 2003). CHFR and RNF8 (A ubiquin ligase) ubiquitinate histones (H2A and H2B) upon ioinizing radiation (Al-Hakim et al., 2010; Wu et al., 2011). These ubiquitinations seem to be important for the eventual activation of the key DNA damage checkpoint effector, ATM (Derks et al., 2006; Lavin and Kozlov, 2007). Recently, CHFR was reported to interact with MAD2, an important component of the spindle assembly checkpoint. CHFR knockdown resulted in mislocalization of MAD2 and disruption of the MAD2/CDC20 interaction. The cysteine-rich region of CHFR appears to be the essential domain for the CHFR/MAD2 interaction and for promoting interaction between MAD2 and CDC20 to inhibit the anaphase-promoting complex (Privette et al., 2008; Keller and Petty, 2011).


    M.musculus 5730484M20Rik RIKEN cDNA 5730484M20 gene, R.norvegicus LOC288734 similar to RIKEN cDNA 5730484M20, budding yeast proteins, Dma1 and Dma2 are 58% identical to each other and are possible homologs of human CHFR. Dma1 and Dma2 have roles in spindle formation and formation of septin ring during cytokinesis (Fraschini et al., 2004).



    No germline mutations have been reported yet.


    A panel of 53 lung carcinomas has been screened with matching normal tissue and 3 mutations were found, one of which was associated with loss of heterozygosity. Mutations found in patient samples were: C587T, G695C (both between the FHA and RING domains) and T1697C (in the C-terminal cysteine rich region of CHFR). However, no correlation was found with a specific diagnosis or stage of the disease in the patients (Mariatos et al., 2003). No clear pathogenic mutations in the CHFR coding sequence have been observed in the analysis of tumors (Privette and Petty, 2008).


    Hypermethylation of the CHFR promoter has been the most commonly reported mechanism lowering CHFR expression observed in tumors (Privitte and Petty, 2008).

    Implicated in

    Entity name
    Gastric cancer
    In gastric cancer, methylation of CHFR promoter is highly recurrent (Hu et al., 2011; Li et al., 2014; Satoh et al., 2003). Decreased CHFR expression has been shown in 20% of gastric cell lines and 39% of primary gastric cancers tested (Satoh et al., 2003). In a study with 102 paraffin-embedded gastric cancer samples, 34% of samples showed methylation. No association was found between methylation of CHFR promoter with gender, age, tumor size, tumor differentiation, and lymph node metastasis. According to Cox proportional hazards model in docetaxel-treated gastric cancer patients, resistance to docetaxel was found in CHFR unmethylated patients. CHFR methylation may serve as a docetaxel-sensitive marker in human gastric cancer (Li et al., 2014).
    Entity name
    Lung cancer
    Loss of detectable CHRF levels has been linked to aberrant hypermethylation in lung cancer (Mizuno et al., 2002). Apart from hypermethylation, inactivation of CHFR gene by missense mutations is reported for lung carcinomas (Mariatos et al., 2003). In a study with 165 lung carcinomas, 10% were found to have hypermethylated CHFR promoter. In addition,
    CHFR hypermethylation was significantly correlated with poor prognosis of lung carcinomas, suggesting a therapeutic potential for CHFR targeted approaches (Koga et al., 2013).
    A lung cancer patient sample demonstrates loss of heterozygosity for a CA repeat located on a BAC that contains the CHFR gene. Several other cancers demonstrate allelic imbalance involving chromosome band 12q24 but specific analysis of CHFR in these samples has not been investigated.
    Entity name
    Breast cancer
    An initial screening resulted with 50% of 24 breast cancer cell lines to have CHFR expression (Erson and Petty, 2004). CHFR protein levels were also low in 36% of breast cancer patients. Lack of CHFR expression in primary cancers was associated with increased tumor size and estrogen receptor negativity (Privette et al., 2007). In another study, 110 primary breast cancers were investigated for methylation status, only 0.9% showed hypermethylation of CHFR promoter (Tokunaga et al., 2006). Although hypermethylation of CHFR promoter is common in various cancers, this study showed it to be a rare event in primary breast carcinomas. Moreover, an interaction between CHFR and PARP-1 was shown to have an important role in cell cycle regulation. CHFR, by its E3 ubiquitin ligase function, caused degradation of PARP-1, which lead to cell cycle arrest in prophase. These findings suggested a novel potential therapeutic approach for combinational chemotherapy with PARP inhibitors for breast cancer cells (Kashima et al., 2012).
    Entity name
    Methylation of CHFR promoter was detected in 39% of leukemia patients. CHFR hypermethylation incidence was shown to be unchanged between acute myelocytic leukemia and acute lymphocytic leukemia (Gong et al., 2005).
    Entity name
    Esophageal cancer
    When expression level of CHFR was investigated, 4 out of 15 esophageal cancer cell lines (26.7%) and 7 out of 43 (16.3%) primary esophageal cancers showed loss of CHFR expression due to hypermethylation of promoter (Shibata et al., 2002). In another study, CHFR transcript was found to be downregulated in 79% of esophageal adenocarcinomas (44 of 56 samples) compared to 41 normal samples. Immunohistochemical analysis also correlated with expression analysis, 75% (56 of 75) of samples showed either weak or no immunostaining. Hypermethylation of promoter correlated with low CHFR expression in esophageal cancer patients; 31% of samples (18 of 58) displayed significant hypermethylation (Soutto et al., 2010). Another recent study used 40 esophageal squamous cell carcinoma patient samples for RT-qPCR analysis of CHFR expression. Aberrant hypermethylation of the CHFR promoter was observed in 13 of 29 primary esophageal cancers. The CHFR expression levels of the methylated samples was significantly lower than that of the unmethylated samples (Suzuki et al., 2014)
    Entity name
    Hepatocellular carcinoma
    Aberrant methylation was detected in 22 of 65 (35%) primary hepatocellular carcinomas (HCC), compared to noncancerous liver cells (Sakai et al., 2005). Also, methylation of CHFR was found to be significantly correlated with advanced disease stage (p=0.037) and an infiltrated growth pattern (p=0.047). In another study with 70 HCC samples, methylation frequency of CHFR was 43% (30 out of 70) (Li et al., 2012). 5-aza-2-deoxycytidine (5-aza-dC) treatment of HCC cell lines restored expression of CHFR.
    Entity name
    Prostate cancer
    In a genome profiling study, blood and bone-marrow samples of prostate cancer patients were investigated using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) (Schwarzenbach et al., 2011). MS-MLPA detected genetic and epigenetic aberrations of 37 tumor suppressor genes including CHFR.
    Entity name
    Head and neck cancer
    19% of 126 head and neck cancer patients showed methylation of a group of tumor suppressors. CHFR was one of the most frequently methylated genes in tumor tissue compared to normal (Yalniz et al., 2011).
    Entity name
    Cervical cancer
    Out of 14 cervical adenocarcinoma specimens tested by methylation-specific PCR, 2 of them (12.3%) showed aberrant methylation of CHFR (Banno et al., 2007). When six cell lines derived from human cervical carcinoma were analyzed, hypermethylaton of CHFR was observed in HeLa and SKG-IIIb cells. In another study, sequential methylation of eight genes including CHFR was linked to HPV-induced cervical carcinogenesis (Henken et al., 2007).


    Pubmed IDLast YearTitleAuthors
    181725002008Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins.Ahel I et al
    210560142010The ubiquitous role of ubiquitin in the DNA damage response.Al-Hakim A et al
    177863012007Epigenetic inactivation of the CHFR gene in cervical cancer contributes to sensitivity to taxanes.Banno K et al
    145620382003The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains.Bothos J et al
    185044342008Chfr interacts and colocalizes with TCTP to the mitotic spindle.Burgess A et al
    249289462014CHFR promoter methylation indicates poor prognosis in stage II microsatellite stable colorectal cancer.Cleven AH et al
    125383482003Frequent hypermethylation of the 5' CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer.Corn PG et al
    171671782006Promoter methylation precedes chromosomal alterations in colorectal cancer development.Derks S et al
    233513072012Primary cilia and aberrant cell signaling in epithelial ovarian cancer.Egeberg DL et al
    146944452004CHFR-associated early G2/M checkpoint defects in breast cancer cells.Erson AE et al
    151460582004Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr.Fraschini R et al
    196903862009Deficiencies in Chfr and Mlh1 synergistically enhance tumor susceptibility in mice.Fu Z et al
    162012592005Methylation of gene CHFR promoter in acute leukemia cells.Gong H et al
    179717712007Sequential gene promoter methylation during HPV-induced cervical carcinogenesis.Henken FE et al
    203009772011Pathobiologic implications of methylation and expression status of Runx3 and CHFR genes in gastric cancer.Hu SL et al
    223378722012CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.Kashima L et al
    234153742013CHFR aberrant methylation involves a subset of human lung adenocarcinoma associated with poor clinical outcomes.Koga T et al
    193260842009Nuclear localization of Chfr is crucial for its checkpoint function.Kwon YE et al
    174570592007ATM activation and DNA damage response.Lavin MF et al
    247485012015Predictive value of CHFR and MLH1 methylation in human gastric cancer.Li Y et al
    218642952012Promoter hypermethylation of DNA damage response genes in hepatocellular carcinoma.Li Z et al
    146125122003Inactivating mutations targeting the chfr mitotic checkpoint gene in human lung cancer.Mariatos G et al
    119484162002Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers.Mizuno K et al
    186334602008CHFR: A Novel Mitotic Checkpoint Protein and Regulator of Tumorigenesis.Privette LM et al
    163347922005Aberrant methylation of the CHFR gene in advanced hepatocellular carcinoma.Sakai M et al
    146951712003Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer.Satoh A et al
    206837292011Genomic profiling of cell-free DNA in blood and bone marrow of prostate cancer patients.Schwarzenbach H et al
    109356422000Chfr defines a mitotic stress checkpoint that delays entry into metaphase.Scolnick DM et al
    123764792002Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer.Shibata Y et al
    240126912013TOPK and PTEN participate in CHFR mediated mitotic checkpoint.Shinde SR et al
    146388682003Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR.Shtivelman E et al
    205641042010Epigenetic and genetic silencing of CHFR in esophageal adenocarcinomas.Soutto M et al
    121216442002Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate.Stavridi ES et al
    156743232005The CHFR mitotic checkpoint protein delays cell cycle progression by excluding Cyclin B1 from the nucleus.Summers MK et al
    243488232014Epigenetic silencing of checkpoint with fork-head associated and ring finger gene expression in esophageal cancer.Suzuki Y et al
    208559742010Alternative efficacy-predicting markers for paclitaxel instead of CHFR in non-small-cell lung cancer.Takeshita M et al
    165020172006Aberrant hypermethylation of the promoter region of the CHFR gene is rare in primary breast cancer.Tokunaga E et al
    128109452003Epigenetic inactivation of CHFR in human tumors.Toyota M et al
    121216422002FHA: a signal transduction domain with diverse specificity and function.Tsai MD et al
    246392832014Association between CHFR methylation and chemosensitivity of paclitaxel in advanced gastric cancer.Wang M et al
    217060082011Chfr and RNF8 synergistically regulate ATM activation.Wu J et al
    208604342011Simultaneous methylation profiling of tumor suppressor genes in head and neck cancer.Yalniz Z et al
    157935872005Chfr is required for tumor suppression and Aurora A regulation.Yu X et al

    Other Information

    Locus ID:

    NCBI: 55743
    MIM: 605209
    HGNC: 20455
    Ensembl: ENSG00000072609


    dbSNP: 55743
    ClinVar: 55743
    TCGA: ENSG00000072609


    Gene IDTranscript IDUniprot

    Expression (GTEx)


    Protein levels (Protein atlas)

    Not detected


    Pubmed IDYearTitleCitations
    181725002008Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins.177
    118070902002The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.55
    128109452003Epigenetic inactivation of CHFR in human tumors.52
    145620382003The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains.39
    153028562004Chfr acts with the p38 stress kinases to block entry to mitosis in mammalian cells.39
    207340642010A large-scale candidate gene association study of age at menarche and age at natural menopause.38
    195840752009Novel susceptibility loci for second primary tumors/recurrence in head and neck cancer patients: large-scale evaluation of genetic variants.36
    223378722012CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.36
    119121572002Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.33
    191827912009Chfr is linked to tumour metastasis through the downregulation of HDAC1.32


    Ayse E Erson-Bensan ; Hesna Begum Akman ; Elizabeth M Petty

    CHFR (Checkpoint with fork-head associated and ring finger)

    Atlas Genet Cytogenet Oncol Haematol. 2014-12-01

    Online version:

    Historical Card

    2004-06-01 CHFR (Checkpoint with fork-head associated and ring finger) by  Ayse E Erson,Elizabeth M Petty 

    Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA (EMP)