CENPH Human

What is CENPH and what is its primary function in human cells?

CENPH, also known as Interphase centromere complex protein 35 (ICEN35), is a protein encoded by the CENPH gene in humans that specifically and constitutively localizes to kinetochores throughout the cell cycle . It functions as a critical component of the CENPA-NAC (nucleosome-associated) complex, which plays a central role in the assembly of kinetochore proteins, mitotic progression, and chromosome segregation . The CENPA-NAC complex is responsible for recruiting the CENPA-CAD (nucleosome distal) complex and may be involved in incorporating newly synthesized CENPA into centromeres . Functionally, CENPH is required for chromosome congression and efficient alignment of chromosomes on the metaphase plate during cell division . Like other centromeric proteins such as CENPA and CENPC, CENPH deficiency results in chromosome missegregation and mitotic failure, highlighting its essential role in maintaining genomic stability .

How does CENPH interact with other centromeric proteins in kinetochore assembly?

CENPH occupies a specific position within the hierarchical assembly of kinetochore proteins, being colocalized with CENPA and CENPC in the inner kinetochores . Studies have demonstrated that CENPH is required for recruiting CENPC and CENP-50 to the kinetochore structure, establishing its role in the early stages of kinetochore assembly . The CENP-C complex that includes CENPH has been characterized as a large polygonal structure that physically associates with four to six CENP-A nucleosomes, as revealed through atomic force microscopy (AFM) and transmission electron microscopy (TEM) . Recent high-resolution analysis of human centromeric chromatin indicates that CENP-C complexes undergo significant structural changes throughout the cell cycle, with a marked increase in height during mitosis compared to G1 and S phases, while maintaining the same Feret's diameter . Furthermore, CENP-C-bound CENP-A nucleosomes adopt a taller configuration compared to unbound CENP-A nucleosomes, suggesting that CENPH and other CCAN components influence the conformation of centromeric chromatin . This protein network plays a critical role in maintaining centromere homeostasis by supporting a unique higher-order structure of centromeric chromatin and regulating its accessibility to transcriptional machinery .

What experimental approaches are used to study CENPH expression and localization?

Researchers employ various experimental techniques to investigate CENPH expression and localization in human cells. Immunofluorescence microscopy using specific anti-CENPH antibodies remains the gold standard for visualizing CENPH localization at kinetochores, typically revealing a punctate nuclear staining pattern . For detecting protein expression levels, Western blot analysis after native chromatin immunoprecipitation (nChIP) has been successfully used to isolate CENPH and associated proteins . Recombinant protein production systems, such as expression in Escherichia coli, provide purified CENPH fragments for in vitro studies, with commercial preparations available for research (e.g., recombinant human CENPH protein fragments expressed in E. coli with >90% purity) . Genetic approaches include cloning full-length CENPH cDNA using RT-PCR with specific primers designed based on the sequence information available in databases . In animal models, particularly zebrafish, CENPH function has been studied through transposon-mediated insertional mutagenesis and morpholino-based knockdown approaches targeting the 5'UTR of the CENPH mRNA . For accurate genotyping in these models, genomic DNA extraction followed by PCR amplification using specific primer combinations has proven effective for identifying wild-type, heterozygous, and homozygous mutants .

What phenotypes result from CENPH deficiency in cellular and animal models?

CENPH deficiency produces severe phenotypes in both cellular and animal models, highlighting its essential function in cell division and development. In cultured human and chicken cells, depletion of CENPH causes chromosome missegregation and ultimately leads to cell death, demonstrating its critical role in maintaining genomic stability . A more comprehensive understanding of CENPH function comes from the zebrafish "stagnant and curly" (stac) mutant, which carries a Tol2 transposon element inserted at the cenph locus . These mutant embryos exhibit discernible cell death as early as 20 hours post-fertilization, followed by extensive apoptosis and morphological abnormalities including an upward curly tail during the pharyngula period and severe deformation around 5 days post-fertilization . The phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, confirming that cenph deficiency is responsible for the observed defects . Mechanistically, the intrinsic apoptosis pathway becomes hyperactivated in stac mutants, and p53 knockdown partially blocks the excess apoptosis, suggesting involvement of p53-dependent pathways . At the cellular level, mitotic cells in stac mutants show chromosome missegregation and typically arrest in G2/M phase, providing direct evidence of CENPH's role in chromosome segregation during mitosis .

How is CENPH expression regulated during development and aging?

The regulation of centromeric proteins, including CENPH and related proteins like CENP-A, undergoes significant changes during development and aging. Studies on CENP-A, a crucial partner of CENPH in the kinetochore, have shown that its expression dramatically declines with age in specific cell types . In human pancreatic beta-cells, CENP-A protein expression exhibits an inverse relationship with age - while 100% of beta-cells in the human fetal pancreas exhibit nuclear staining for CENP-A in a punctuate pattern, this expression declines rapidly with age and becomes essentially undetectable by age 30 . This age-dependent decline appears to be tissue-specific, as exocrine pancreatic cells continue to express CENP-A at approximately the same level from ages 18-45 . The mechanism underlying this decline seems to be post-transcriptional, as there is no correlation between CENP-A mRNA levels and age . While direct studies on CENPH expression during aging are more limited, its functional relationship with CENP-A suggests potentially similar regulatory mechanisms. In mouse models, the decline in centromeric protein expression with age is less pronounced than in humans, indicating species-specific differences in the regulation of these proteins . These findings have significant implications for understanding age-related changes in cell proliferation capacity and the development of age-associated disorders.

What methodologies are most effective for manipulating CENPH expression in experimental settings?

Manipulating CENPH expression in experimental settings requires careful selection of techniques based on the specific research objectives. For knockdown experiments, antisense morpholinos have proven effective, particularly in zebrafish models where two different morpholinos (cenph-MO1: 5′-TCCAATCCTGTCTGAAACTGCCGCC-3′ and cenph-MO2: 5′-GTTACTGGAACTCATCTTTGTATGT-3′) targeting the 5'UTR of cenph have been successfully employed . These morpholinos, when injected into one-cell embryos, effectively reduce CENPH expression and recapitulate the phenotypes observed in genetic mutants . For overexpression studies, mRNA synthesis in vitro from linearized plasmids using the mMessage Machine kit has been used to generate CENPH mRNA for microinjection . The full-length coding sequence of cenph can be amplified by RT-PCR with specific primers (e.g., cph5RNA: 5′-CGGAATTCCACCATGAGTTCCAGTAACGTTAATC-3′ and cph3RNA: 5′-CCCTCGAGTTAGCTGGGTATGTGTTCCAGTTTC-3′) . In established cell lines, transient transfection approaches using lipid-based reagents like Lipofectamine 2000 have been successful for introducing CENPH expression constructs . For stable cell lines, lentiviral or retroviral delivery systems may offer advantages for long-term expression studies. To analyze the consequences of CENPH manipulation, researchers commonly employ a combination of techniques including immunofluorescence microscopy for localization studies, flow cytometry for cell cycle analysis, and biochemical assays such as co-immunoprecipitation to examine protein-protein interactions within the kinetochore complex .

How does CENPH contribute to centromeric chromatin structure and function?

CENPH contributes significantly to centromeric chromatin structure and function through its role in the CENP-C complex. High-resolution analysis of human centromeric chromatin has revealed that CENP-C complex-bound chromatin shows distinct structural properties, being notably refractory to MNase digestion, which suggests a more compact or protected chromatin state . Atomic force microscopy studies have demonstrated that the CENP-C complex, which includes CENPH, increases in height throughout the cell cycle, reaching its maximum dimensions during mitosis . This physical change in the complex coordinates with functional transitions in centromeric chromatin. Furthermore, research has identified two distinct CENP-A nucleosomal configurations within human centromeres: a shorter variant and a taller variant that associates with the CENP-C complex . This relationship suggests that CENPH, as part of the CENP-C complex, contributes to altering the conformation of CENP-A nucleosomes, potentially affecting chromatin accessibility and function . The CENP-C complex appears to regulate centromere homeostasis by supporting a unique higher-order structure of centromeric chromatin and modulating its accessibility to transcriptional machinery . Different models have been proposed for centromeric chromatin folding, including a boustrophedon arrangement in chicken chromatin when exposed to low-salt buffers and a looping model for point centromeres, though the precise endogenous structure in human cells remains under investigation . These findings collectively indicate that CENPH plays a crucial role in organizing centromeric chromatin into functional structures required for proper kinetochore assembly and chromosome segregation.

How can researchers effectively isolate and analyze CENPH-associated protein complexes?

The isolation and analysis of CENPH-associated protein complexes require sophisticated biochemical approaches tailored to preserve the integrity of these delicate structures. Native chromatin immunoprecipitation (nChIP) has emerged as a powerful technique for purifying endogenous centromeric chromatin associated with the CENP-C complex, which includes CENPH . This approach involves careful cell lysis under conditions that maintain native protein-protein and protein-DNA interactions, followed by immunoprecipitation using antibodies specific to centromeric proteins . Serial nChIP, where sequential immunoprecipitations target different components (e.g., first CENP-C, then CENP-A), has proven especially valuable for isolating specific subcomplexes with high purity . Western blot analysis after nChIP can confirm the presence of various CCAN components within the isolated complexes . For structural analysis of these complexes, a combination of atomic force microscopy (AFM), immuno-AFM, and transmission electron microscopy (TEM) has been successfully employed to characterize their dimensions and configurations . These techniques have revealed that the CENP-C complex is a large polygonal structure physically associated with multiple CENP-A nucleosomes . For functional studies, researchers can combine these isolation techniques with biochemical assays such as MNase digestion to assess chromatin accessibility, or with mass spectrometry for comprehensive protein identification and quantification . To study dynamic changes in these complexes, synchronization of cells at different stages of the cell cycle (G1, S phase, and mitosis) followed by complex isolation and analysis has proven informative in revealing cell cycle-dependent structural alterations .

What are the current challenges in developing research tools for studying CENPH interactions and dynamics?

Developing effective research tools for studying CENPH interactions and dynamics faces several significant challenges that researchers must navigate. One primary challenge lies in preserving the native conformation and interactions of CENPH during experimental manipulations. The protein exists within a complex network of kinetochore components, and disruption of these interactions can lead to artifacts that misrepresent its true functions . Current antibody-based approaches for visualizing or isolating CENPH-containing complexes may not capture the full spectrum of interactions or may alter the dynamics of these assemblies . Another challenge involves reconciling results from in vitro reconstituted systems with in vivo observations. While recent cryo-EM work has revealed structures of in vitro reconstituted budding yeast and human inner kinetochore components, the endogenous kinetochore structure in living human cells remains less defined . The dynamic nature of kinetochore assembly throughout the cell cycle further complicates this research, requiring temporal resolution in addition to spatial detection capabilities . Developing tools for studying CENPH in specific contexts, such as during aging or in disease states, presents additional challenges, particularly given the tissue-specific regulation observed with related proteins like CENP-A . Technical limitations in visualizing individual protein molecules within the densely packed kinetochore structure make it difficult to track CENPH dynamics in real-time during mitosis. Advanced tools combining genetic engineering approaches (such as CRISPR-Cas9 for endogenous tagging) with super-resolution microscopy and quantitative proteomics will be necessary to overcome these challenges and gain deeper insights into CENPH biology.

What protocols exist for expressing and purifying recombinant CENPH for in vitro studies?

Recombinant CENPH protein production relies on established protocols that can be optimized for specific experimental requirements. Expression of human CENPH in Escherichia coli represents a widely used approach, with commercial preparations available that achieve >90% purity suitable for a range of biochemical applications . These systems typically utilize a fragment-based approach, expressing defined regions of the protein rather than the full-length version due to challenges with solubility and stability of the complete protein . For instance, a human CENPH fragment spanning amino acids 136 to 247 has been successfully expressed in E. coli with excellent purity . These recombinant proteins are commonly engineered with affinity tags such as hexahistidine (His6) tags to facilitate purification through metal affinity chromatography, resulting in constructs with sequences such as "MGSSHHHHHHS SGLVPRGSHM..." . For functional studies, researchers must consider whether denatured or native conformations are required; protocols exist for both formats, though denatured preparations are often used for applications like SDS-PAGE and antibody production, while native preparations are essential for interaction studies . The purification process typically involves bacterial cell lysis under appropriate buffer conditions, followed by affinity chromatography, and potentially additional purification steps such as size exclusion or ion exchange chromatography to achieve higher purity when needed. Quality control assessments using SDS-PAGE, Western blotting, and mass spectrometry confirm the identity and purity of the final preparation before use in downstream applications like protein-protein interaction studies, enzymatic assays, or structural analyses.

How can researchers effectively measure CENPH localization and dynamics throughout the cell cycle?

Studying CENPH localization and dynamics throughout the cell cycle requires a multifaceted approach combining cell synchronization with advanced imaging and biochemical techniques. Cell synchronization methods, including thymidine block for G1/S boundary arrest or nocodazole treatment for mitotic arrest, provide populations of cells at defined cell cycle stages for comparative analysis . Immunofluorescence microscopy with anti-CENPH antibodies allows visualization of the protein's distribution, typically revealing a punctate pattern at kinetochores that can be quantified for signal intensity and spatial distribution . For higher resolution assessment, super-resolution microscopy techniques such as structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), or photoactivated localization microscopy (PALM) offer nanometer-scale precision in locating CENPH within the kinetochore structure . Live-cell imaging approaches using fluorescently tagged CENPH (e.g., CENPH-GFP fusion proteins) enable real-time tracking of protein dynamics during mitotic progression . For biochemical analysis, native chromatin immunoprecipitation (nChIP) followed by Western blotting allows quantitative assessment of CENPH levels in chromatin-associated complexes at different cell cycle stages . This approach has revealed that the CENP-C complex, which includes CENPH, increases in height throughout the cell cycle, reaching a maximum during mitosis . Combined immunoprecipitation and mass spectrometry approaches can identify cell cycle-specific interaction partners of CENPH, providing insights into the dynamic assembly and function of kinetochore structures . Finally, fluorescence recovery after photobleaching (FRAP) experiments with fluorescently tagged CENPH can measure the protein's turnover rate within kinetochores, revealing whether it remains stably associated or undergoes exchange with a soluble pool during different cell cycle phases.

What analytical methods are used to assess the functional consequences of CENPH mutations or depletion?

Assessing the functional consequences of CENPH mutations or depletion requires a comprehensive analytical toolkit that spans molecular, cellular, and organismal approaches. At the cellular level, researchers employ fluorescence microscopy to visualize chromosome alignment and segregation defects, typically revealing misaligned chromosomes and lagging chromosomes during anaphase in cells with CENPH dysfunction . Quantitative analysis of mitotic progression through live-cell imaging with phase contrast or fluorescent chromosomal markers provides temporal information on mitotic delays or arrests . Flow cytometry for DNA content analysis can reveal the accumulation of cells in G2/M phase, a characteristic finding in CENPH-deficient systems . Molecular analyses include assessment of apoptotic pathway activation through Western blotting for cleaved caspase proteins or TUNEL assays to detect DNA fragmentation, as CENPH deficiency often triggers apoptotic cell death . For organism-level studies in model systems like zebrafish, researchers evaluate developmental progression, morphological abnormalities, and survival rates, with CENPH-deficient embryos typically exhibiting severe developmental defects and early mortality . Specific phenotypes associated with CENPH deficiency in zebrafish include discernible cell death as early as 20 hours post-fertilization, extensive apoptosis, upward curly tail, and severe deformation . Rescue experiments, where wild-type CENPH mRNA is introduced into deficient systems, provide powerful evidence of phenotype specificity . Similarly, phenocopy experiments using antisense morpholinos to knock down CENPH in wild-type backgrounds can confirm that the observed defects are directly attributable to CENPH deficiency rather than secondary effects . Finally, genetic interaction studies, where CENPH manipulations are combined with alterations in other pathways (such as p53 knockdown), can illuminate the mechanisms through which CENPH deficiency exerts its effects .

How does CENPH research contribute to our understanding of chromosomal instability in cancer?

CENPH research provides critical insights into the mechanisms of chromosomal instability in cancer, a hallmark feature of many aggressive tumors. Studies have demonstrated that both overexpression and depletion of CENPH can lead to chromosome missegregation, highlighting the protein's pivotal role in maintaining genomic stability . CENPH overexpression in human HCT116 cells and mouse 3T3 cells induces aneuploidy due to chromosome missegregation, potentially resulting from abnormal localization of ectopic CENPH within the kinetochore structure . This mechanistic understanding helps explain the observation that CENPH is upregulated in several types of human tumors, suggesting a potential role in promoting genomic instability during cancer development . Paradoxically, research in zebrafish models has shown that heterozygous stac fish (with CENPH deficiency) develop invasive tumors at a dramatically reduced rate compared to wild-type siblings, indicating a potential tumor-suppressive effect of partial CENPH reduction . This finding parallels observations with other kinetochore proteins, where altered levels have been shown to affect cancer susceptibility in animal models . The complex relationship between CENPH expression and cancer risk likely reflects the principle that proper chromosome segregation requires precisely balanced levels of kinetochore proteins - both excess and insufficiency can disrupt this balance, but with different consequences for cell viability and transformation . These observations contribute to a deeper understanding of how kinetochore dysfunction contributes to chromosomal instability in cancer and may inform the development of novel therapeutic approaches targeting mitotic processes in cancer cells.

What implications does age-dependent regulation of centromeric proteins have for regenerative medicine?

The age-dependent regulation of centromeric proteins, including CENPH and CENP-A, has profound implications for regenerative medicine, particularly in tissues where cell replication is critical for repair and renewal. Studies have shown that CENP-A, a protein required for chromosome segregation in mitosis, declines precipitously with age in an islet-specific manner in humans, becoming essentially undetectable after age 29, while exocrine pancreatic cells retain CENP-A expression . This tissue-specific loss of essential mitotic machinery helps explain the dramatic decline in beta-cell replication with age, a phenomenon that significantly impacts diabetes treatment strategies seeking to expand beta-cell mass . The mechanism appears to be post-transcriptional, as there is no correlation between CENP-A mRNA levels and age, suggesting opportunities for intervention at the protein level . The decline is more pronounced in humans than in mice, highlighting important species differences that must be considered when translating findings from animal models to human applications . These observations suggest that strategies aimed at restoring centromeric protein expression in aged tissues might enhance regenerative capacity by removing a fundamental block to cell division . For regenerative medicine approaches that rely on expanding specific cell populations either in vivo or in vitro, understanding and potentially manipulating the age-related decline in centromeric proteins could significantly improve outcomes . Furthermore, this knowledge informs the development of more effective cell-based therapies by identifying optimal donor cell ages or pretreatment strategies to enhance replicative potential.