Affinage

CENPX

Centromere protein X · UniProt A8MT69

Length
81 aa
Mass
9.0 kDa
Annotated
2026-06-09
38 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CENPX (CENP-X/MHF2) is a small histone-fold protein that operates at the interface of kinetochore assembly and DNA repair through a single conserved heterodimerization partner, CENP-S (MHF1) (PMID:19620631, PMID:20347429). At centromeres, CENP-X is a constitutive kinetochore component whose loss reduces outer kinetochore plate size and increases intrakinetochore distance, establishing it as essential for stable outer kinetochore assembly (PMID:19620631); with CENP-S it integrates into the larger CENP-T-W-S-X complex that preferentially engages ~100 bp of linker DNA, forms a (CENP-T-W-S-X)2 assembly, and induces positive DNA supercoils, with the DNA-binding and supercoiling activity residing in the CENP-T/CENP-W subunits rather than CENP-S/X (PMID:24234442). CENP-S/X assembles de novo at centromeres during S and G2 phase, with CENP-X exchanging far more rapidly than CENP-S, and the complex is required for spindle assembly checkpoint signaling and for kinetochore loading of CCAN components (PMID:24522885, PMID:36537249). In its second role, the CENP-S/CENP-X (MHF1/MHF2) heterodimer associates with the FANCM helicase to form a histone-fold tetramer that binds branched DNA—preferring forks and four-way junctions—and stimulates FANCM branch migration activity, with the structural basis defined by crystal structures of the MHF tetramer alone, bound to FANCM, and bound to DNA (PMID:20347429, PMID:22510687, PMID:24390579). Through this activity CENP-X promotes FANCD2 monoubiquitination and chromatin loading of the Fanconi anemia core complex (PMID:20347429), and is recruited to double-strand breaks in an RSF1- and ATM-dependent manner where it promotes XRCC4 loading and non-homologous end-joining while being dispensable for homologous recombination (PMID:23974106, PMID:24800743). At centromere DNA repeats it acts with FANCM to suppress crossovers and gross chromosomal rearrangements (PMID:28977643).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2009 High

    Established CENP-X as a bona fide constitutive kinetochore protein with a defined structural role, answering whether it contributes to kinetochore architecture.

    Evidence Genetic knockouts in chicken DT40 and siRNA in HeLa cells with live imaging and electron microscopy of the outer plate

    PMID:19620631

    Open questions at the time
    • Did not define the biochemical activity of the CENP-S/X dimer
    • Mechanism linking CENP-X loss to outer plate shrinkage unresolved
  2. 2010 High

    Revealed CENP-X has a second identity as MHF2, partnering with MHF1/CENP-S to bind DNA and stimulate FANCM branch migration, connecting it to the Fanconi anemia pathway.

    Evidence Co-IP, protein stability and DNA-binding assays, branch migration assay, siRNA with MMC/CPT sensitivity in cells

    PMID:20347429

    Open questions at the time
    • Structural basis of FANCM engagement not yet defined
    • DNA substrate preference not yet mapped
  3. 2012 High

    Defined at atomic resolution how the MHF1-MHF2 tetramer forms and engages FANCM, establishing the architectural plasticity underlying FANCM recruitment.

    Evidence X-ray crystallography of MHF1-MHF2 alone and with FANCM fragment, plus in vivo localization; complemented by yeast structural/genetic analysis of the heterotetramer interface

    PMID:22325783 PMID:22510687

    Open questions at the time
    • Did not capture the DNA-bound state
    • Functional consequences of supercoiling vs branch migration not separated
  4. 2013 High

    Distinguished the centromeric CENP-T-W-S-X complex's biochemical behavior, showing it binds linker DNA and induces positive supercoils with the activity carried by CENP-T/W rather than CENP-S/X.

    Evidence In vitro DNA-binding and supercoiling assays with mutagenesis and kinetochore targeting assays

    PMID:24234442

    Open questions at the time
    • Specific contribution of CENP-X to complex stability not isolated
    • Functional role of positive supercoiling in vivo unresolved
  5. 2013 Medium

    Placed CENP-S/X within the DSB repair pathway, showing RSF1-dependent assembly that promotes NHEJ via XRCC4 while being dispensable for HR.

    Evidence siRNA, laser microirradiation, NHEJ/HR repair assays; SAXS analysis of the (MHF1-MHF2)4 octamer DNA-binding surface; fission yeast genetics separating repair from centromere functions and demonstrating centromere-repeat crossover suppression

    PMID:23886707 PMID:23974106 PMID:24026537 PMID:28977643

    Open questions at the time
    • Single-lab pathway placement for NHEJ
    • Mechanism by which CENP-S/X directs XRCC4 loading unknown
    • SAXS surface patch not validated by mutagenesis
  6. 2014 High

    Defined the upstream recruitment signal and DNA recognition mode for CENP-X at breaks, showing ATM-RSF1-dependent recruitment that drives FANCD2/FANCI monoubiquitination and a structural basis for branched-DNA sensing.

    Evidence Co-IP and laser microirradiation with ubiquitination assays; crystal structure of MHF-DNA complex with biochemical and yeast genetic validation; quantitative live-cell imaging of de novo centromere assembly by FCCS/FRET/FRAP

    PMID:24390579 PMID:24522885 PMID:24800743

    Open questions at the time
    • How ATM/RSF1 selectivity for chromatin sites is achieved unresolved
    • Coupling between branched-DNA binding and FA ubiquitination not mechanistically closed
  7. 2019 Medium

    Extended MHF-FANCM function to replication termination, showing the interaction suppresses inter-fork strand annealing.

    Evidence Fission yeast C-terminal domain mutants with epistasis and in vitro regressed-fork restoration assay

    PMID:31855181

    Open questions at the time
    • Demonstrated in yeast Fml1, human relevance not confirmed
    • Direct role of CENP-X in fork restoration not isolated
  8. 2021 Medium

    Showed the FANCM-MHF interaction is conditional, dissociating under oxidative conditions while the MHF dimer persists, hinting at a regulatable switch.

    Evidence X-ray crystallography of the chicken FANCM-MHF1-MHF2 tripartite complex under varied conditions

    PMID:33439149

    Open questions at the time
    • Physiological trigger for dissociation not established
    • No mutagenesis validation of the conditional interface
  9. 2023 Medium

    Connected the centromeric histone-fold pair to spindle assembly checkpoint control and CCAN/Aurora B localization, broadening CENP-X's mitotic role beyond static architecture.

    Evidence Fission yeast deletion mutants with live imaging and immunofluorescence of SAC and CCAN components

    PMID:36537249

    Open questions at the time
    • Mechanism linking Mhf2 loss to Ark1 mislocalization unknown
    • Human CENP-X role in SAC not directly tested
  10. 2025 Medium

    Quantified the timing of CENP-X at DSBs, placing it during early chromatin remodeling and pathway choice across all interphase stages.

    Evidence Live-cell microirradiation with quantitative recruitment/removal kinetics and cell-cycle phase analysis

    PMID:40450933

    Open questions at the time
    • No functional validation of the observed timing
    • Whether removal kinetics are causally linked to RPA/RAD51 loading not tested

Open questions

Synthesis pass · forward-looking unresolved questions
  • How CENP-X is partitioned between its centromeric and DNA-repair pools, and whether the conditional FANCM dissociation regulates this switch in human cells, remains unresolved.
  • No mechanism for routing CENP-S/X between kinetochore and DSB pools
  • Regulatory triggers for FANCM release not defined in vivo

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 4 GO:0060090 molecular adaptor activity 3 GO:0005198 structural molecule activity 2
Localization
GO:0000228 nuclear chromosome 3 GO:0005634 nucleus 2 GO:0005694 chromosome 2
Pathway
R-HSA-1640170 Cell Cycle 3 R-HSA-73894 DNA Repair 3 R-HSA-4839726 Chromatin organization 2
Partners
CENPSCENPTCENPWFANCMRSF1
Complex memberships
CENP-S/CENP-X (MHF1-MHF2) heterodimerCENP-T-W-S-X complexFANCM-MHF complexkinetochore (CCAN)

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 CENP-X was identified as a new constitutive kinetochore protein forming a subcomplex with CENP-S. CENP-X-deficient chicken DT40 and human HeLa cells show abnormal mitotic behavior, and kinetochore localization of CENP-X requires CENP-T or CENP-K. Loss of CENP-X results in a significant reduction in outer kinetochore plate size and increased intrakinetochore distance, demonstrating its essential role in stable outer kinetochore assembly. Genetic knockouts in chicken DT40 cells, siRNA knockdown in HeLa cells, live cell imaging, electron microscopy of kinetochore outer plate The Journal of cell biology High 19620631
2010 MHF2 (CENP-X) and MHF1 (CENP-S) form a histone-fold-containing heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. Suppression of MHF1 destabilizes FANCM and MHF2, impairs FANCD2 monoubiquitination and foci formation, causes defective chromatin localization of FA core complex proteins, and increases MMC-induced chromosome aberrations. Co-immunoprecipitation, protein stability assays, DNA-binding biochemical assays, branch migration activity assay, siRNA knockdown with cellular phenotyping (immunofluorescence, MMC/CPT sensitivity) Molecular cell High 20347429
2012 Crystal structure of the MHF1-MHF2 (CENP-S/CENP-X) complex reveals that they form a compact tetramer. FANCM binds this tetramer through a 'dual-V' shaped structure, and FANCM-F together with (MHF1-MHF2)2 constitutes a new DNA-binding site coupled to the canonical L1L2 region. Perturbation of MHF-FANCM structural plasticity alters FANCM localization in vivo. X-ray crystallography of MHF1-MHF2 alone and in complex with FANCM fragment, in vivo localization assays Nature communications High 22510687
2012 The yeast MHF complex (Mhf1-Mhf2) adopts a histone-fold architecture structurally similar to the (H3-H4)2 heterotetramer. The heterotetrameric assembly is essential for the function of the complex in DNA repair, as shown by genetic data with interface mutants. X-ray crystallography, yeast genetics (mutant analysis of heterotetramer interfaces) Structure (London, England : 1993) High 22325783
2013 The CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA (not nucleosome-bound DNA), forms a (CENP-T-W-S-X)2 structure, and induces positive DNA supercoils in contrast to canonical nucleosomes. DNA-binding regions in CENP-T or CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling activity and kinetochore targeting of the complex. DNA-binding assays, DNA supercoiling assay, mutagenesis of DNA-binding regions, kinetochore targeting assays Nucleic acids research High 24234442
2013 RSF1 facilitates the assembly of CENP-S and CENP-X at sites of DNA damage. Upon incorporation by RSF1, CENP-S and CENP-X promote assembly of the NHEJ factor XRCC4 at damaged chromatin, thereby promoting non-homologous end-joining. CENP-S and CENP-X are dispensable for homologous recombination. siRNA knockdown, laser microirradiation, immunofluorescence at DSB sites, NHEJ and HR repair assays Cell cycle (Georgetown, Tex.) Medium 23974106
2014 RSF1, in an ATM-dependent manner, recruits CENP-X (MHF2) and CENP-S (MHF1) to DSB sites. CENP-X/MHF2 and CENP-S/MHF1 in turn regulate mono-ubiquitination of FANCD2 and FANCI at DSBs. The ATM-RSF1 interaction is dependent on DSBs and ATM kinase activity. Co-immunoprecipitation, laser microirradiation/immunofluorescence, siRNA knockdown, FANCD2/FANCI ubiquitination assays PLoS biology Medium 24800743
2014 Crystal structure of human MHF1-MHF2 (CENP-S/CENP-X) bound to DNA reveals that MHF senses branched DNA by engaging two duplex arms simultaneously. Biochemical analyses confirm MHF preferentially binds DNA forks and four-way junctions over dsDNA. Mutations at the observed DNA-binding interface reduce cellular resistance to DNA damage. X-ray crystallography of MHF-DNA complex, DNA-binding biochemical assays, yeast genetic experiments with DNA-binding interface mutants Nature communications High 24390579
2014 CENP-S and CENP-X assemble de novo at centromeres during S phase and G2, increasing approximately 3-4 fold in abundance. FRET and fluorescence cross-correlation spectroscopy show that CENP-S and CENP-X exist in complex in soluble form and at centromeres. CENPX exchanges ~10 times faster than CENP-S at centromeres (t1/2 ~10 min vs ~120 min). CENP-T was identified as a strong CENP-S binding partner at centromeres by fluorescent two-hybrid and FRET, forming a CENP-S/X/T-containing complex near histone H3 but not CENP-A. Fluorescence cross-correlation spectroscopy, FRET, conditional labeling (EdU pulse-chase), FRAP, fluorescent two-hybrid assay Open biology High 24522885
2013 In fission yeast, Mhf1 and Mhf2 (CENP-S/CENP-X orthologs) perform two distinct functions: DNA repair/recombination (dependent on interaction with the FANCM ortholog Fml1) and centromere localization for chromosome segregation (independent of Fml1). Together with Fml1, they process sister chromatid junctions to aid chromosome segregation; the Mus81-Eme1 endonuclease acts as a failsafe for unresolved junctions. Fission yeast genetics (deletion mutants, epistasis), chromosome segregation assays, centromere localization experiments Open biology Medium 24026537
2013 A SAXS study revealed that the (MHF1-MHF2)4 octamer presents a long, positively charged patch on its surface that plays a critical role in double-stranded DNA binding, providing the structural basis for anchoring the MHF-FANCM complex to chromatin. Small angle X-ray scattering (SAXS) in combination with crystallographic data FEBS letters Medium 23886707
2017 In fission yeast, Mhf1/CENP-S and Mhf2/CENP-X histone-fold proteins together with Fml1/FANCM helicase suppress crossovers between centromere DNA repeats and prevent gross chromosomal rearrangements mediated by centromere repeats during mitosis. Fission yeast genetics (deletion mutants), recombination assays at centromere and non-centromere loci, chromosome rearrangement assays Nucleic acids research Medium 28977643
2019 The Fml1 (FANCM ortholog) interaction with Mhf1-Mhf2 via its C-terminal domain is required for suppression of inter-fork strand annealing (IFSA) during DNA replication termination. Fml1 can catalyze regressed fork restoration in vitro, providing a plausible mechanism for IFSA suppression. Fission yeast genetics (C-terminal domain mutants, epistasis), in vitro regressed fork restoration assay eLife Medium 31855181
2021 Crystal structure of the chicken FANCM (MHF interaction region)-MHF1-MHF2 tripartite complex reveals that FANCM-MHF interaction involves a mixture of hydrophobic/hydrophilic interactions. Under oxidative conditions or in the presence of MPD, FANCM dissociates from MHF while MHF retains its complexed form, indicating the conditional nature of the FANCM-MHF interaction. X-ray crystallography of tripartite complex, crystallization under various conditions Acta crystallographica. Section F, Structural biology communications Medium 33439149
2023 In fission yeast, Mhf1-Mhf2 (CENP-S/CENP-X counterparts) regulate the spindle assembly checkpoint (SAC). Loss of Mhf2 attenuates the SAC, impairs kinetochore localization of most CCAN components, and alters localization of the Aurora B kinase homolog Ark1 at the kinetochore. Live-cell microscopy, yeast genetics (deletion mutants), immunofluorescence of SAC components and CCAN proteins Journal of cell science Medium 36537249
2025 CENP-X is recruited to DNA double-strand breaks in live HeLa cells with a half-time of ~100 s and removed with a half-time of ~2000 s. Recruitment occurs in G1, S, and G2 phases, with delayed but stronger recruitment in G2. CENP-X recruitment occurs simultaneously with CENP-S, immediately after ATM activation and RNF8-RNF168 activity, and removal coincides with RPA loading and RAD51 assembly, placing CENP-X at DSBs during early chromatin remodeling, pathway choice, and resection. Live-cell microirradiation, quantitative fluorescence microscopy, cell cycle phase analysis, integration with published DDR factor timelines DNA repair Medium 40450933

Source papers

Stage 0 corpus · 38 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2010 MHF1-MHF2, a histone-fold-containing protein complex, participates in the Fanconi anemia pathway via FANCM. Molecular cell 166 20347429
2011 The ABCs of CENPs. Chromosoma 164 21751032
2009 The CENP-S complex is essential for the stable assembly of outer kinetochore structure. The Journal of cell biology 127 19620631
2017 FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres. Proceedings of the National Academy of Sciences of the United States of America 125 28673972
2014 FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors, limit meiotic crossovers. Nucleic acids research 84 25038251
2013 The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA. Nucleic acids research 65 24234442
2012 The structure of the FANCM-MHF complex reveals physical features for functional assembly. Nature communications 58 22510687
2013 Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining. Cell cycle (Georgetown, Tex.) 53 23974106
2014 The RSF1 histone-remodelling factor facilitates DNA double-strand break repair by recruiting centromeric and Fanconi Anaemia proteins. PLoS biology 40 24800743
2012 Rad5-dependent DNA repair functions of the Saccharomyces cerevisiae FANCM protein homolog Mph1. The Journal of biological chemistry 29 22696213
2013 MHF1-2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination. Open biology 28 24026537
2014 A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle. Open biology 24 24522885
2012 A prototypical Fanconi anemia pathway in lower eukaryotes? Cell cycle (Georgetown, Tex.) 24 22895051
2014 The MHF complex senses branched DNA by binding a pair of crossover DNA duplexes. Nature communications 23 24390579
2012 Saccharomyces cerevisiae MHF complex structurally resembles the histones (H3-H4)₂ heterotetramer and functions as a heterotetramer. Structure (London, England : 1993) 22 22325783
2023 The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination. Nucleic acids research 20 36652992
2024 Where do the Fluorine Atoms Go in Inorganic-Oxide Fluorinations? A Fluorooxoborate Illustration under Terahertz Light. Angewandte Chemie (International ed. in English) 18 38344870
2017 Regulation of mitotic recombination between DNA repeats in centromeres. Nucleic acids research 18 28977643
2014 Identifying novel oncogenes: a machine learning approach. Interdisciplinary sciences, computational life sciences 17 24402816
2013 Genetic and physical interactions between the yeast ELG1 gene and orthologs of the Fanconi anemia pathway. Cell cycle (Georgetown, Tex.) 14 23624835
2013 Emerging roles for centromere-associated proteins in DNA repair and genetic recombination. Biochemical Society transactions 13 24256282
2024 KHSRP has oncogenic functions and regulates the expression and alternative splicing of DNA repair genes in breast cancer MDA-MB-231 cells. Scientific reports 12 38926398
2019 The Fml1-MHF complex suppresses inter-fork strand annealing in fission yeast. eLife 9 31855181
2014 Structural analysis of in silico mutant experiments of human inner-kinetochore structure. Bio Systems 7 25451768
2010 Stabilizing and remodeling the blocked DNA replication fork: anchoring FANCM and the Fanconi anemia damage response. Molecular cell 6 20347418
2024 Somatic gene mutations involved in DNA damage response/Fanconi anemia signaling are tissue- and cell-type specific in human solid tumors. Frontiers in medicine 5 39421870
2013 Structural peculiarities of the (MHF1-MHF2)4 octamer provide a long DNA binding patch to anchor the MHF-FANCM complex to chromatin: a solution SAXS study. FEBS letters 5 23886707
2025 Stage-specific phenotypic and transcriptional alterations in HaCaT keratinocytes exposed to acute and chronic blue light. Photochemistry and photobiology 4 40191980
2023 The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation. Journal of cell science 4 36537249
2023 FANCM interacts with the MHF1-MHF2 complex to limit crossover frequency during rice meiosis. The Plant journal : for cell and molecular biology 4 37632767
2022 Comprehensive Analysis of Centromere Protein Family Member Genes in Lung Adenocarcinoma. Critical reviews in eukaryotic gene expression 4 35695666
2019 Therapeutic Silencing of Centromere Protein X Ameliorates Hyperglycemia in Zebrafish and Mouse Models of Type 2 Diabetes Mellitus. Frontiers in genetics 4 31417608
2012 Formation of a centromere-specific chromatin structure. Epigenetics 3 22617165
2021 Structural analysis of the chicken FANCM-MHF complex and its stability. Acta crystallographica. Section F, Structural biology communications 2 33439149
2025 Dynamics of chromatin factors RSF1, CENPS and CENPX at DNA damage sites. DNA repair 1 40450933
2016 Biochemical and Structural Analysis of Kinetochore Histone-Fold Complexes. Methods in molecular biology (Clifton, N.J.) 1 27193847
2025 Towards identification of a holocentromere marker in the lepidopteran model Spodoptera frugiperda. Chromosoma 0 40067534
2024 [Homozygous Variant of FANCM of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism]. Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 0 38948269

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