CENPA Human

What is CENP-A and what is its primary role in human cells?

CENP-A is a histone H3 variant with approximately 48% sequence identity to canonical histone H3, containing a highly diverged N-terminal tail that lacks many well-characterized histone modification sites including H3K4, H3K9, and H3K27 . As the critical factor determining kinetochore positions on each chromosome, CENP-A epigenetically defines the centromere location regardless of the underlying DNA sequence .

CENP-A functions as the foundation of centromeric chromatin by replacing histone H3 in a subset of nucleosomes specifically at active centromeres, where it forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA . This specialized chromatin structure serves as the platform for kinetochore assembly, which is essential for accurate chromosome segregation during mitosis .

How is CENP-A different from canonical histone H3 in terms of structure and dynamics?

CENP-A exhibits the greatest sequence divergence among histone H3 variants, with only 48% similarity to canonical histone H3 . Key structural differences include:

Unlike canonical histone H3, CENP-A is not loaded in conjunction with DNA replication. In human cells, CENP-A loading occurs specifically during G1 phase and requires the specialized chaperone HJURP . This unique loading pattern enables the precise maintenance of centromere identity across multiple cell divisions.

How is CENP-A position maintained through DNA replication?

CENP-A positioning exhibits remarkable precision in maintenance through DNA replication. Recent studies using the Telomere-to-Telomere (T2T) genome assembly, which contains the first complete human centromere sequences, demonstrated that CENP-A molecules deposited in G1 phase are maintained in their exact position through DNA replication .

Despite the dilution of CENP-A during DNA replication (which occurs without concurrent CENP-A synthesis), the protein is precisely reloaded onto the same sequences within daughter centromeres, thereby maintaining unique centromere identity among human cells . This precise maintenance involves:

• Recognition of existing CENP-A nucleosomes by the Mis18 licensing complex

• Recruitment of HJURP to these sites

• Deposition of new CENP-A precisely at the original locations in the subsequent G1 phase

This mechanism ensures the faithful inheritance of centromere position independently of DNA sequence, representing a true epigenetic inheritance system .

What methodologies are most effective for studying CENP-A positioning in human centromeres?

Several complementary methodologies have proven effective for studying CENP-A positioning:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • Allows genome-wide mapping of CENP-A binding sites

  • Can be coupled with the T2T genome assembly for precise mapping to repetitive centromeric regions

  • Requires high-quality antibodies specific to CENP-A

• SNAP-tag or CLIP-tag technologies:

  • Enable pulse-chase experiments to track old versus newly synthesized CENP-A

  • Allow visualization of CENP-A dynamics throughout the cell cycle

  • Can distinguish generational cohorts of CENP-A proteins

• Atomic Force Microscopy:

  • Provides structural insights into reconstituted CENP-A nucleosomes

  • Shows "beads on a string" images similar to canonical nucleosomes

• DNA-RNA Immunoprecipitation (DRIP):

  • Captures DNA-RNA hybrids in their native chromosomal context

  • Can be followed by qPCR amplification of centromere-specific alpha-satellites

• Centromeric Chromosome-Orientation Fluorescence In Situ Hybridization (Cen-CO-FISH):

  • Monitors recombination at centromeres

  • Effective for tracking centromere stability and rearrangements

These methods have collectively advanced our understanding of how CENP-A positioning is established and maintained with remarkable precision through multiple cell divisions.

How does CENP-A depletion affect DNA replication and genome stability?

CENP-A depletion during S phase has profound effects on DNA replication and genome stability. Research shows that rapid removal of CENP-A specifically in S phase (but not other cell cycle stages) leads to:

• Replication Fork Progression Issues:

  • Impaired DNA replication progression at centromeres

  • Accumulation of R-loops with increased centromeric transcripts

• DNA Recombination Events:

  • Increased recombination at alpha-satellite repeats

  • R-loop-dependent recombination events

• Mitotic Abnormalities:

  • Unfinished replication leading to anaphase bridges

  • Chromosome breakage specifically at centromeric regions

• Structural Chromosomal Aberrations:

  • Centromere breakage and fragmentation

  • Formation of isochromosomes

  • High proportion of whole-arm chromosome translocations involving both acrocentric and metacentric chromosomes

Experimental evidence demonstrates that CENP-A depletion upon release from G1 arrest, S-phase entry, or early S-phase arrest triggers severe centromere rearrangements, while depletion in G2 or metaphase-arrested cells does not cause such aberrations . This indicates that CENP-A plays a crucial role in maintaining genomic stability specifically during DNA replication.

What is the relationship between CENP-A and R-loop formation at centromeres?

CENP-A plays a critical role in preventing excessive R-loop formation at centromeres during DNA replication. R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced single-stranded DNA.

Research using DNA-RNA immunoprecipitation (DRIP) analysis has revealed:

• CENP-A removal leads to a reproducible increase in centromere-associated R-loops, particularly in late S phase when most centromeres are replicated

• These R-loops are specifically associated with centromeric alpha-satellite regions, as shown by qPCR amplification of centromere X-specific alpha-satellites and pan-centromeric alpha-satellites

• The R-loop signals are specific to DNA-RNA hybrids since they:

  • Are not detectable with control immunoglobulin (IgG)

  • Are eliminated by treatment with RNase H1 (which specifically degrades RNA in RNA-DNA hybrids)

• Immunofluorescence-based detection using S9.6 antibody confirms induction of centromere-associated DNA-RNA hybrids upon acute CENP-A depletion during S phase, but not when CENP-A is depleted during G2 or M phase

These findings indicate that CENP-A chromatin plays an essential role in preventing R-loop accumulation during centromere replication, thereby safeguarding centromere integrity and chromosomal stability.

How do CENP-A nucleosomes oscillate throughout the cell cycle?

CENP-A nucleosomes undergo unique structural oscillations throughout the cell cycle in human cells. This phenomenon has been observed in parallel studies in both human cells and budding yeast . The oscillations involve:

• Structural Transitions:

  • CENP-A nucleosomes may transition between different structural states (octamers, hexamers, homotypic tetramers, and heterotypic tetramers) during different phases of the cell cycle

  • These transitions likely reflect the dynamic nature of centromeric chromatin and its adaptation to different cellular processes

• Loading Dynamics:

  • Unlike canonical histones, CENP-A loading occurs exclusively during G1 phase in human cells

  • This loading is mediated by the CENP-A-specific chaperone HJURP and requires the Mis18 licensing complex

• Inheritance Pattern:

  • Pre-existing CENP-A is equally distributed to daughter strands during DNA replication

  • New CENP-A is loaded during the subsequent G1 phase at precisely the same locations

These oscillations likely serve critical biological functions, including:

• Facilitating the assembly of the kinetochore during mitosis

• Enabling the precise epigenetic inheritance of centromere identity

• Accommodating the structural demands of DNA replication and transcription at centromeres

What methodological approaches can distinguish between different CENP-A nucleosomal species?

Investigating the different structural states of CENP-A nucleosomes requires sophisticated methodological approaches:

• Biochemical Reconstitution:

  • In vitro nucleosome assembly using purified components

  • Can be mediated by nucleosome assembly protein-1 (NAP-1)

  • Atomic force microscopy visualization of "beads on a string" structures

• DNase I Digestion Assays:

  • Generation of DNA ladders with repeats of approximately 10 bp

  • Indicates DNA wrapping around protein complexes

  • Can reveal structural differences between canonical and CENP-A nucleosomes

• Glycerol Gradient Sedimentation:

  • Isolation of mononucleosomes

  • Determination of molecular mass (~200 kDa for octameric complexes)

  • Analysis of DNA and protein composition

• Cross-linking Mass Spectrometry:

  • Captures transient protein-protein interactions

  • Can detect structural transitions between different nucleosomal states

• FRET (Förster Resonance Energy Transfer):

  • Measures distances between fluorescently labeled components

  • Can detect conformational changes in CENP-A nucleosomes during the cell cycle

• Cryo-electron Microscopy:

  • High-resolution structural analysis of different CENP-A nucleosomal states

  • Can reveal atomic-level details of various conformations

These methodologies collectively provide insights into the dynamic nature of CENP-A nucleosomes and their structural transitions throughout the cell cycle.

What are the most effective systems for controlled CENP-A depletion in research?

Several experimental systems have proven effective for controlled CENP-A depletion:

• Auxin-Inducible Degron (AID) System:

  • Allows rapid and reversible protein degradation

  • Can achieve CENP-A depletion within hours

  • Enables cell cycle stage-specific depletion

  • Minimizes compensatory mechanisms that may occur with slower depletion methods

• Small Molecule-Induced Protein Degradation:

  • Includes PROTAC (Proteolysis Targeting Chimera) technology

  • Allows dose-dependent control of CENP-A levels

  • Can be combined with cell cycle synchronization techniques

• Conditional Gene Knockout Systems:

  • Cre-lox recombination for genomic deletion

  • Tet-on/off systems for transcriptional control

  • Allow longer-term studies of CENP-A depletion effects

• RNAi and CRISPR Interference:

  • siRNA or shRNA for temporary knockdown

  • CRISPRi for targeted transcriptional repression

  • Useful for partial depletion studies

For time-sensitive experiments, research has demonstrated that CENP-A depletion can be effectively combined with cell cycle synchronization:

• G1 arrest using CDK4/6 inhibitor Palbociclib

• S-phase arrest using thymidine block

• G2/M arrest using specific inhibitors

These approaches allow researchers to deplete CENP-A at specific cell cycle stages to determine when CENP-A function is most critical.

How can researchers effectively distinguish CENP-A epialleles in different human cell lines?

CENP-A epialleles (different epigenetic states at the same genetic locus) have been observed at several centromeres in different human cell lines . To effectively distinguish these epialleles:

• Mapping to the T2T Assembly:

  • The Telomere-to-Telomere (T2T) genome assembly containing complete human centromere sequences provides a valuable resource for precise CENP-A mapping

  • ChIP-seq data can be aligned to this reference to identify cell line-specific patterns

• Single-Cell Technologies:

  • Single-cell ChIP-seq or CUT&Tag can reveal heterogeneity within populations

  • Allows detection of distinct CENP-A positioning patterns in individual cells

• Clonal Analysis:

  • Establishing and analyzing multiple clones from the same cell line

  • Comparing CENP-A positioning between clones after multiple cell divisions

  • Can reveal the stability of epialleles across generations

• Comparative Analysis Across Cell Types:

  • Systematic comparison of CENP-A binding patterns across different human cell lines

  • Identifies conserved and variable regions of CENP-A enrichment

  • Helps distinguish inherent biological variation from technical artifacts

• Functional Validation:

  • Correlating CENP-A positioning with kinetochore recruitment sites

  • Testing the functional consequences of different CENP-A epialleles on chromosome segregation

These approaches can help researchers understand the natural variation in CENP-A positioning among human cells while ensuring that experimental findings are not confounded by cell line-specific differences.