CENPB Human
Description
Molecular Structure and Characteristics
CENPB Human is a DNA-binding protein derived from transposases of the pogo DNA transposon family . It features a molecular weight of approximately 65-68 kDa in its native form, though recombinant versions with tags may reach around 80 kDa . The protein contains several functionally important domains:
DNA Binding Domain
The DNA binding capability of CENPB resides within its NH2-terminal 125-amino acid region, which contains four potential alpha-helices . This region features a helix-loop-helix DNA binding motif that specifically recognizes the CENP-B box, a 17-bp sequence (YTTCGTTGGAARCGGGA) found in centromeric repetitive DNA . The binding affinity is significantly reduced when the CENP-B box is methylated, indicating epigenetic regulation of CENPB binding .
Dimerization Domain
CENPB contains a dimerization domain at its C-terminus that enables the formation of protein dimers . Chemical cross-linking studies have confirmed that CENPB forms a dimer in solution . This dimerization is functionally significant as it allows CENPB to juxtapose two distant CENP-B boxes, thereby creating higher-order structures in the centromere .
Amino Acid Sequence
The full amino acid sequence of recombinant human CENPB includes an N-terminal His-tag followed by the native sequence. The recombinant protein typically includes approximately 600-623 amino acids, with additional tags accounting for the higher observed molecular weight compared to the native protein .
Functional Role in Centromere Formation
CENPB serves critical functions in centromere organization and kinetochore assembly:
CENP-B Box Binding
The primary function of CENPB involves its specific binding to the CENP-B box sequence in centromeric repetitive DNA . This binding is preferential to unmethylated CENP-B boxes located in the centromere dip region (CDR), areas characterized by reduced CpG methylation . The alphoid DNA-CENPB complex formed was the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes .
Higher-Order Structure Organization
Through both DNA-protein and protein-protein interactions, CENPB organizes a higher-order structure in the centromere . The dimerization capability allows CENPB to bundle two distant CENP-B boxes, creating structural foundations for centromere assembly . CENPB binds directly to both CENP-A and CENP-C, further stabilizing centromere structure .
Kinetochore Assembly
CENPB contributes significantly to kinetochore assembly by organizing arrays of centromere satellite DNA into higher-order structures . The unmethylated CENP-B boxes serve as broad-based anchors in centromeric DNA, where binding of dimeric CENPB triggers the attachment of CENP-A, leading to stable assembly of CENP-C and additional inner and outer kinetochore components .
Distribution of CENP-B Boxes in Human Chromosomes
Recent research based on the Telomere-to-Telomere (T2T) genome project has revealed the distribution pattern of CENP-B boxes across human chromosomes . While the number of CENP-B boxes varies significantly between chromosomes, their density remains relatively consistent:
| Chromosome | Chromosome Size (Mb) | Centromere Span (Mb) | CDR Span (Kb) | Number of CENP-B Boxes in CDR | Density (number/Kb) |
|---|---|---|---|---|---|
| 1 | 249 | 30 | 307 | 880 | 2.87 |
| 2 | 242 | 14 | 172 | 378 | 2.20 |
| 3 | 198 | 16 | 211 | 500 | 2.37 |
| 4 | 190 | 15 | 260 | 643 | 2.47 |
| 5 | 182 | 13 | 214 | 616 | 2.88 |
| 6 | 171 | 13 | 157 | 335 | 2.13 |
| 7 | 159 | 13 | 296 | 748 | 2.53 |
| 8 | 145 | 12 | 132 | 360 | 2.73 |
| 9 | 138 | 42 | 147 | 385 | 2.62 |
| 10 | 134 | 12 | 278 | 802 | 2.88 |
| 11 | 135 | 13 | 249 | 509 | 2.04 |
| 12 | 133 | 13 | 284 | 800 | 2.82 |
| 13 | 114 | 23 | 216 | 563 | 2.61 |
| 14 | 107 | 18 | 195 | 559 | 2.87 |
| 15 | 102 | 23 | 239 | 742 | 3.10 |
| 16 | 90 | 26 | 157 | 430 | 2.74 |
| 17 | 83 | 14 | 172 | 505 | 2.94 |
| 18 | 80 | 15 | 170 | 500 | 2.94 |
| 19 | 59 | 15 | 424 | 1158 | 2.73 |
| 20 | 64 | 17 | 249 | 635 | 2.55 |
| 21 | 47 | 17 | 145 | 338 | 2.33 |
| 22 | 51 | 21 | 190 | 522 | 2.75 |
| X | 156 | 13 | 158 | 288 | 1.82 |
The data reveals that while the number of CENP-B boxes varies 4-fold between chromosomes (from 288 in chromosome X to 1158 in chromosome 19), their density varies less than 2-fold, ranging from 1.82 in chromosome X to 3.10 in chromosome 15, with a mean density of 2.61 ± 0.33 number/Kb . This narrow density range is functionally significant as it ensures a uniform pull of the spindle on the centromeres during cell division .
Production and Characteristics of Recombinant CENPB
Recombinant CENPB protein is produced for various research and diagnostic applications:
Expression Systems
Human CENPB is commonly expressed in either:
Recombinant Protein Characteristics
The recombinant CENPB typically features:
Clinical Significance and Autoimmunity
CENPB has substantial clinical relevance, particularly in autoimmune conditions:
CENPB as an Autoantigen
CENPB represents one of the most important centromeric autoantigens . It is a target of autoantibodies known as anti-centromere antibodies, which are detected in various autoimmune conditions . The protein-antibody interaction involves CENPB binding with IgG-type human auto-antibodies .
Association with CREST Syndrome
Anti-CENPB antibodies are present in the sera of up to 80% of patients with CREST syndrome, a variant of systemic sclerosis characterized by calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia . This high prevalence makes CENPB an important diagnostic marker for this condition.
Diagnostic Applications
Recombinant CENPB is utilized in various diagnostic applications including:
CENPB in Cancer Pathology
Recent research has implicated CENPB in cancer biology, particularly in hepatocellular carcinoma:
Role in Hepatocellular Carcinoma
Studies analyzing TCGA and GEO datasets have revealed higher expression of CENPB mRNA in hepatocellular carcinoma (HCC) tissues compared to normal tissues . CENPB expression increases with advancing pathological stage and histological grade, suggesting a role in cancer progression .
Prognostic Value
Functional Role in Cancer Progression
Experimental studies involving knockdown of CENPB in HCC cell lines (Hep3B and MHCC97) resulted in significant inhibition of cell proliferation and invasion, suggesting an oncogenic function . Bioinformatics analysis identified miR-29a as a potential negative regulator of CENPB expression, which was validated through dual-luciferase reporter assays .
Current Research and Future Perspectives
Research on CENPB continues to evolve, with several key areas of focus:
Chromosomal Stability Studies
Recent investigations have found that alpha-satellite arrays with more CENP-B boxes and thus greater CENP-B binding were less likely to mis-segregate during cell division . In CENP-A depleted RPE-1 cells, a statistically significant negative correlation exists between the rate of chromosome mis-segregation and the abundance of CENP-B boxes . This highlights the importance of CENPB in maintaining chromosomal stability.
Evolutionary Conservation
CENPB is highly conserved across mammalian species, suggesting its fundamental importance in centromere function . Comparative studies between humans, great apes, and New World monkeys have revealed variations in CENP-B box locations within centromeric DNA repeat units, potentially associated with higher-order repeat structures of centromeric DNA .
Therapeutic Potential
The discovery of CENPB's role in HCC progression suggests potential therapeutic applications . The relationship between miR-29a and CENPB expression offers a possible avenue for targeted therapies . Future research may focus on developing interventions that modulate CENPB expression or function in cancer cells.
Product Specs
Q&A
What is the role of CENP-B in centromere function and kinetochore assembly?
CENP-B is a DNA-binding protein that specifically interacts with a 17-base pair sequence known as the CENP-B box within α-satellite DNA. Its primary role is to stabilize centromeric chromatin by facilitating the assembly of CENP-A, a histone H3 variant essential for centromere identity and function. CENP-B also interacts with CENP-C, a key nucleator of kinetochore assembly, thereby ensuring proper chromosome segregation during cell division .
The mechanism involves two critical steps: (1) the recruitment of histone chaperones and chromatin modifiers by CENP-B to establish an open chromatin state conducive to CENP-A deposition, and (2) the stabilization of kinetochore components through direct protein-protein interactions. Experimental studies have shown that mutations in the CENP-B box or depletion of CENP-B result in reduced centromeric levels of CENP-C and increased rates of chromosome mis-segregation .
How does the density and distribution of CENP-B boxes influence centromere stability?
The density of CENP-B boxes within α-satellite DNA is a critical determinant of centromere stability. Studies have revealed that while the number of CENP-B boxes varies significantly between chromosomes, their density (number per kilobase) remains relatively uniform, with a mean value of approximately 2.61 ± 0.33 boxes per kilobase . This uniformity ensures consistent microtubule attachment forces across centromeres.
The precise localization of kinetochore attachment sites within centromeres has been mapped using epigenetic markers such as hypomethylated CpG dinucleotides within the centromere dip region (CDR). These regions are enriched in unmethylated CENP-B boxes, which serve as high-affinity binding sites for CENP-B. The narrow density range of these boxes supports even spindle microtubule dynamics, thereby reducing the likelihood of chromosome mis-segregation .
What experimental approaches are used to study the interaction between CENP-B and other centromeric proteins?
Several experimental techniques are employed to investigate the interactions between CENP-B and other centromeric proteins:
-
Chromatin Immunoprecipitation (ChIP): This method is used to identify DNA sequences bound by CENP-B in vivo. By coupling ChIP with sequencing (ChIP-seq), researchers can map the distribution of CENP-B across centromeres.
-
Protein Interaction Assays: Techniques such as co-immunoprecipitation (Co-IP) and yeast two-hybrid assays are utilized to study direct interactions between CENP-B and proteins like CENP-A and CENP-C.
-
CUT&RUN: A targeted chromatin profiling technique that combines antibody-guided cleavage with sequencing to localize proteins like CENP-A and CENP-B at specific genomic regions.
-
Gene Editing: CRISPR/Cas9-mediated knockout or mutation of the CENP-B gene allows researchers to assess its functional role in centromere assembly and kinetochore formation.
-
Fluorescence Microscopy: High-resolution imaging techniques such as super-resolution microscopy are used to visualize the spatial organization of centromeric proteins in live or fixed cells .
How do epigenetic modifications affect the binding affinity of CENP-B?
Epigenetic modifications, particularly DNA methylation at CpG sites within the CENP-B box, significantly influence the binding affinity of CENP-B. Unmethylated CpG dinucleotides within the box provide high-affinity binding sites for CENP-B, facilitating its role in kinetochore assembly. In contrast, methylation reduces binding affinity nearly to nonspecific levels, impairing kinetochore formation.
Recent studies utilizing epigenetic mapping techniques have identified hypomethylated regions within centromeres, termed centromere dip regions (CDRs), which coincide with enriched binding sites for both CENP-A and CENP-B. These findings underscore the functional relevance of epigenetic regulation in maintaining centromere integrity .
What are human artificial chromosomes (HACs), and how does CENP-B contribute to their formation?
Human artificial chromosomes (HACs) are engineered chromosomes designed to replicate and segregate independently within host cells. They serve as valuable tools for studying chromosome biology and gene therapy applications.
CENP-B plays a pivotal role in HAC formation by promoting de novo assembly of functional centromeres on transfected α-satellite DNA containing intact CENP-B boxes. The presence of these boxes is essential for recruiting histone chaperones, chromatin modifiers, and kinetochore components necessary for HAC stability. Mutations in the CENP-B box or depletion of CENP-B result in diminished HAC formation efficiency .
How does the absence or mutation of CENP-B affect chromosome segregation?
In mouse models, similar phenotypes have been observed, indicating that the role of CENP-B in supporting centromere function via maintenance of centromeric components is conserved across species .
What computational tools are available for analyzing α-satellite DNA sequences containing CENP-B boxes?
Several bioinformatics tools have been developed for analyzing α-satellite DNA sequences:
-
RepeatMasker: Identifies repetitive DNA elements, including α-satellites, within genomic sequences.
-
MEME Suite: Detects conserved motifs such as the 17-bp CENP-B box within DNA sequences.
-
Genome Browsers: Tools like UCSC Genome Browser allow visualization of α-satellite regions annotated with epigenetic data.
-
Custom Scripts: Researchers often develop Python or R scripts for analyzing sequence features such as CpG methylation patterns or box density.
These tools enable detailed characterization of α-satellite DNA regions critical for understanding their role in centromere function .
How do histone modifications influence the recruitment of chromatin modifiers by CENP-B?
CENP-B recruits various chromatin modifiers through its acidic domain, which interacts with histone modifications such as H3K9 trimethylation (H3K9me3) and H3K36 methylation (H3K36me). These modifications play distinct roles:
-
H3K9me3: Enriched in heterochromatic regions flanking centromeres; it recruits heterochromatin protein 1 (HP1), contributing to chromosome stability.
-
H3K36me: Facilitates open chromatin states conducive to de novo assembly of functional centromeres containing CENP-A.
The interplay between these histone modifications ensures a balance between heterochromatic repression and euchromatic activation at centromeres .
Can variations in α-satellite DNA composition affect kinetochore positioning?
Yes, variations in α-satellite DNA composition can influence kinetochore positioning by altering the distribution and density of functional elements like the CENP-B box. Recent advances using T2T-CHM13 maps have provided high-resolution insights into these variations across human chromosomes.
For example, while some chromosomes exhibit a four-fold variation in the number of CENP-B boxes within their centromeres, their density remains relatively constant across chromosomes. This consistency is crucial for maintaining uniform spindle attachment forces during cell division .
Product Science Overview
Centromere Protein B is a DNA-binding protein derived from transposases of the pogo DNA transposon family . It contains two main functional domains:
- Helix-Loop-Helix DNA Binding Motif: Located at the N-terminus, this domain allows CENP-B to recognize and bind to a specific 17-base pair sequence known as the CENP-B box in the centromeric alpha satellite DNA .
- Dimerization Domain: Located at the C-terminus, this domain facilitates the dimerization of the protein, which is essential for its function in centromere formation .
The primary function of CENP-B is to organize arrays of centromere satellite DNA into a higher-order structure, which then directs the formation of centromeres and the assembly of kinetochores on mammalian chromosomes . This organization is critical for the proper segregation of chromosomes during cell division.
Centromere Protein B is also known as a major centromere autoantigen. It is recognized by sera from patients with anti-centromere antibodies, which are often present in autoimmune diseases such as CREST syndrome and Raynaud’s disease . Additionally, CENP-B has been identified as a potential biomarker for small-cell lung cancer .
Recombinant Centromere Protein B is produced using recombinant DNA technology, which involves inserting the CENPB gene into an expression system to produce the protein in vitro. This recombinant protein is used in various research applications, including the study of centromere structure and function, as well as in diagnostic assays for autoimmune diseases .
Research on CENP-B has provided significant insights into the mechanisms of chromosome segregation and the role of centromeres in maintaining genomic stability. The recombinant form of CENP-B is particularly valuable in these studies, as it allows for detailed biochemical and structural analyses.
