Centromere Protein B Research Articles

CENPB promotes the proliferation of hepatocellular carcinoma and is directly regulated by miR-29a.

Hepatocellular carcinoma (HCC) is a significant global health concern as it ranks as the sixth most common malignant tumor and the third leading cause of cancer-related deaths. In this study, we analyzed the expression of centromere protein B (CENPB) mRNA in HCC using TCGA and GEO datasets. Immunohistochemistry (IHC) was performed to determine CENPB protein levels in 490 HCC patients. Our findings revealed higher expression of CENPB mRNA in HCC tissues across the three datasets. Additionally, as the pathological stage and histological grade advanced, CENPB expression increased. Patients with elevated levels of CENPB mRNA and protein demonstrated shorter overall survival (OS) and recurrence-free survival (OS). Notably, CENPB protein showed prognostic value in patients with stage I/II, AFP levels below 400 ng/ml, and tumor size less than 5 cm. Using multivariate regression analysis in 490 HCC patients, we developed nomograms to predict 1-year, 3-year, and 5-year OS and RFS. Knockdown of CENPB in Hep3B and MHCC97 cell lines resulted in significant inhibition of cell proliferation and invasion. Furthermore, bioinformatics analysis identified miR-29a as a potential negative regulator of CENPB expression, which was validated through a dual-luciferase reporter assay. In conclusion, our findings suggest that CENPB may serve as an oncogenic factor in HCC and is directly regulated by miR-29a, highlighting its potential as a promising therapeutic target.

Cytotoxic effect of combining two antisense oligonucleotides against telomerase rna component (hTR and mRNA of centromere protein B (CENP-B) in hepatocellular carcinoma cells.

Telomerase is a ribonucleoprotein enzyme that plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation and helping in the high rate proliferation of cancer cells. Our study is designed to evaluate the effect of using combined antisense oligonucleotides (ASOs) targeting (hTR) and mRNA of CENP-B on liver cancer cells. Compared with a single treatment, combination treatment with Locked Nucleic Acid (LNA) ASO (hTR) and (CENP-B) (6.25 nM from each) exhibit the maximum synergistic cytotoxic effect. hTR and CENP-B mRNA was abrogated while hTERT expression was disappeared. Caspase-3, Bax, and Bcl-2 were not detected, indicating caspase-independent cell death. A significant reduction in [Tumor necrosis factor (TNF-α) and Transforming growth factor (TGF-β)] coincides with elevation in Nitric oxide (NO) secretions was observed. Taken together; our data suggest that combination treatment with LNA ASO (hTR) and (CENP-B) could provide a promising strategy for cancer treatment by controlling many pathways concurrently. This might open a new prospective application of antisense in cancer therapy.

Anticentromere antibody induced by immunization with centromere protein and Freund\u2019s complete adjuvant may interfere with mouse early-stage embryo

BackgroundAnticentromere antibody (ACA) is a member of the antinuclear antibody spectrum (ANAs) which has been speculated to be associated with subfertility. Thus, the present study aimed to investigate the induction of ACA production and its potential interference with early-stage embryos.MethodsRecombinant centromere protein-A (CENP-A) or centromere protein-B (CENP-B) and complete Freund’s adjuvant (CFA) were used to immunize mice. Serum ACA level was then evaluated by using an indirect immunofluorescence test. Immunofluorescence assay was performed to detect IgG in follicles in ovarian tissues and early-stage embryos.ResultsFollowing treatment, serum positive ACA was observed in mice treated with CENP and CFA. Furthermore, IgG were detected in follicular fluid and early-stage embryos from mice treated with CENP and CFA.ConclusionsThis study preliminarily indicated that ACA induced by CENP and CFA may penetrate into the living embryos of early-stage in mice.

Massive parallel sequencing in a family with rectal cancer

BackgroundWe have previously reported a family with a suspected autosomal dominant rectal and gastric cancer syndrome without any obvious causative genetic variant. Here, we focused the study on a potentially isolated rectal cancer syndrome in this family.MethodsWe included seven family members (six obligate carriers). Whole-exome sequencing and whole-genome sequencing data were analyzed and filtered for shared coding and splicing sequence and structural variants among the affected individuals.ResultsWhen considering family members with rectal cancer or advanced adenomas as affected, we found six new potentially cancer-associated variants in the genes CENPB, ZBTB20, CLINK, LRRC26, TRPM1, and NPEPL1. All variants were missense variants and none of the genes have previously been linked to inherited rectal cancer. No structural variant was found.ConclusionBy massive parallel sequencing in a family suspected of carrying a highly penetrant rectal cancer predisposing genetic variant, we found six genetic missense variants with a potential connection to the rectal cancer in this family. One of them could be a high-risk genetic variant, or one or more of them could be low risk variants. The p.(Glu438Lys) variant in the CENPB gene was found to be of particular interest. The CENPB protein binds DNA and helps form centromeres during mitosis. It is involved in the WNT signaling pathway, which is critical for colorectal cancer development and its role in inherited rectal cancer needs to be further examined.

Abstract PO055: A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides

Abstract Background: Current cancer treatment tactics ought to be selective to carcinogenic cells and non-destructive to normal cells. Telomerase is a ribonucleoprotein enzyme which plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. While it is not found in somatic cells, its reactivation is obligatory in malignant cells to maintain the length of the telomere and prevent its senescence. Antisense-based strategies against human Telomerase RNA component (hTR) led to cancer cell death at some time after administering of antisense oligonucleotide (ASOs), and this lag time is one of the major drawbacks for this approach in treating cancer. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation. CENP-B is highly expressed in cancer cells helping in high rate proliferation. Materials and Methods: HepG2 cells were transfected with several concentrations (6.25, 12.5 and 25 nM) of LNA ASO against hTR and CENP-B mRNA either individually or in combination. MTT assay was used to assess the Hep-G2 cytotoxicity 24 and 48 hrs post-transfection. Telomerase activity, hTR, and CENP-B mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Effect of antisense treatment on Caspase-3, Bax, and Bcl-2 was also investigated by RT-PCR. Transforming growth factor (TGF-β), Tumor necrosis factor (TNF-α) and, Nitric oxide (NO) were estimated in supernatant collected 48 after transfection with ASOs by enzyme-linked immunosorbent assay (ELISA). Results: Our study conclusively demonstrates that using a single treatment with LNA ASO- hTR or CENP-B led to dose and time-dependent reduction in HepG2 cell viability. The combination of two LNA antisense (hTR + CENP-B) with different concentrations showed a synergistic cytotoxic effect. LNA ASO treatments (6.25 nM from each) exhibit the greatest synergistic cytotoxic effect. hTR and CENP-B mRNA in HepG2 cells were totally abrogated. Telomerase activity was disappeared. Expression of Caspase-3, Bax, and Bcl-2 was not detected after transfecting cells with LNA ASO hTR alone or in-combination with LNA ASO CENP-B indicates to caspase-independent cell death. A significant decrease in TGF-β and TNF-α and elevation in NO secretions was observed. Conclusion: LNA ASO hTR is potent in decreasing telomerase activity with a rapid anti-tumor effect with Caspase-independent cell death. This tumor inhibitory effect was magnified by ASO CENP-B indicating to the synergistic cytotoxic effect of oligonucleotides combination at certain concentrations. The rapid inhibitory effects of combined oligonucleotides on tumor growth open a new prospective application of antisense in cancer therapy. Citation Format: Ahmed Mohamed El-Desoky, Yasser Ali, Roba Talaat. A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO055.

Significance of anti-tubulin-α-1C autoantibody in systemic sclerosis

OBJECTIVE To detect the serum level of a novel autoantibody, anti-tubulin-α-1C, in patients with systemic sclerosis (SSc) and to investigate its clinical significance. METHODS Anti-tubulin-α-1C antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in 62 patients with SSc, 38 systemic lupus erythematosus (SLE), 24 primary Sjogren's syndrome (pSS) patients, and 30 healthy controls (HCs). Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), immunoglobulin A(IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), C3, C4, rheumatoid factor (RF), antinuclear antibody(ANA), anti-centromere antibodies(ACA), anticardiolipin (aCL), anti-dsDNA antibody, anti-Sm antibody, anti-RNP antibody, anti-Scl-70 antibody, anti-Ro52 antibody, anti-SSA antibody, anti-SSB antibody, centromere protein A(CENP-A), centromere protein B (CENP-B) were measured by standard laboratory techniques. Raynaud's phenomenon and modified Rodnan skin score(MRSS) were recorded to evaluate the disease status of SSc. Independent sample t test, Chi square test, Mann-Whitney U test, Spearman rank correlation were used for statistical analyses. RESULTS The serum anti-tubulin-α-1C antibody concentration in SSc group was 81.24±34.38, the serum anti-tubulin-α-1C antibody concentration in SLE group was 87.84±38.52, the serum anti-tubulin-α-1C antibody concentration in pSS group was 59.79±25.24, and the serum anti-tubulin-α-1C antibody concentration in healthy group was 39.37±18.7. Multivariate analysis revealed that anti-tubulin-α-1C antibody levels were significantly increased in the SSc and SLE patients. The expression level of anti-tubulin-α-1C antibody in SSc was higher compared with the pSS group and the health control group (P < 0.01). Further analysis demonstrated that the elevated anti-tubulin-α-1C antibody were correlated with the SSc inflammation and disease activity markers ESR(r=0.313, P=0.019), The levels of anti-tubulin-α-1C antibody were also significantly correlated with MRSS(r=0.636, P < 0.01). The best cut-off value for the diagnose of SSc was 76.77 as mean+2SD value. The proportion of Raynaud's phenomenon was higher in the group of anti-tubulin-α-1C autoantibody-postive SSc patients than that in anti-tubulin-α-1C autoantibody negative group(71.4% vs. 37.5%, P=0.039). The proportions of anti-Scl-70 antibody, anti-CENP antibody and anti-cardiolipin antibody were higher in the group of anti-tubulin-α-1C autoantibody-postive SSc patients than in the anti-tubulin-α-1C autoantibody negative group (37.9% vs. 15.2%, 34.5% vs. 12.1%, 13.8 vs. 0, respectively, all P < 0.05). CONCLUSION Based on this explorative stu-dy, the level of anti-tubulin-α-1C antibody increased in the serum of the patients with SSc. There were correlations between anti-tubulin-α-1C autoantibody and clinical and laboratory indicators of the SSc patients. It may become a novel biomarker indicative of active SSc and could be applied in future clinical practice.

Pathogenesis of degenerative dementia: Emerging role for inflammation

AbstractBackgroundRecent evidence proposes a role for immune‐mediated mechanisms in pathogenesis of neurodegenerative dementias. Contribution of inflammatory processes to the progression of neurodegeneration has also been hypothesized. While deposition of various proteins is recognised as the pathological hallmark for the neurodegenerative dementias, Alzheimer’s disease (AD) and Frontotemporal dementia (FTD), the role of inflammation secondary to immune dysregulation in neurodegeneration remains to be determined. In this study, we aimed to investigate the profile of autoantibodies in patients diagnosed to have neurodegenerative dementia and compare the association between the different subtypes.MethodWe performed immunological screening in a large cohort of neurodegenerative dementias. Diagnosis was based on standard criteria, on review of clinical, MR imaging and laboratory features. Dementia types comprised of Alzheimer’s disease (AD), Frontotemporal dementia (FTD), and Dementia with Lewy Bodies (DLB). The immunological screening consisted of antinuclear antibody (ANA) profile, paraneoplastic and autoimmune encephalitis profile.ResultOf 274 consecutive patients with degenerative dementia evaluated in the Cognitive Disorders Clinic (AD = 62, FTD = 202, DLB = 10), autoantibody profile was done in 174(63.5%). ANA profile was positive in 18.3%, paraneoplastic panel in 8.3% and autoimmune panel in 2.1% of patients. We observed a significantly higher frequency of immunopositivity in patients with FTD (34.4%) compared to AD(10.4%) and DLBD(14.2%). The range of antibodies detected included antibodies against anti‐Ro 52(SS‐A), anti‐La(SS‐B), PM‐Scl, centromere protein B(CENP‐B), dsDNA, histones and ribosomal P‐proteins in the ANA profile; anti‐Hu, anti‐Ri, anti‐Yo, anti‐CV‐2, PNMA2 (Ma2/Ta), Anti‐amphiphysin, Anti‐ SOX1, anti‐glial nuclear antibody(AGNA), anti‐Tr, Anti‐GAD65, anti‐Zic4, anti‐titin and anti‐recoverin in paraneoplastic and in the autoimmune profile autoantibodies present were N‐methyl‐D‐asparate(NMDA), alpha‐amino‐3‐hydroxy‐5 methyl‐4‐isoxazoleproprionic acid (AMPA), Leucine rich glioma‐inactivated protein‐1(LGI1), Contactin‐associated protein‐2(CASPR2), and GABAB receptor antibody. There were no significant systemic complaints in majority of patients, and no difference was observed in the mean age, gender and duration of illness between immune‐positive and immune‐negative patients.ConclusionOur findings further substantiate the emerging role for neuroinflammation secondary to immune dysregulation, in patients with classical neurodegenerative dementias. Establishing the profile of specific autoantibodies that occur in association with dementia subtypes, especially FTD, may aid in understanding mechanisms that underlie role of autoimmunity in neurodegenerative dementias, with therapeutic implications.

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

1-Methylpyrene induces chromosome loss and mitotic apparatus damage in a Chinese hamster V79-derived cell line expressing both human CYP1A2 and sulfotransferase 1A1

1-Methylpyrene (1-MP) is a ubiquitous environmental pollutant and rodent carcinogen. Its mutagenic activity depends on sequential activation by various CYP and sulfotransferase (SULT) enzymes. Previously we have observed induction of micronuclei and mitotic arrest by 1-MP in a Chinese hamster (V79)-derived cell line expressing both human CYP1A2 and SULT1A1 (V79-hCYP1A2-hSULT1A1), however, the mode of chromosome damage and the involvement of mitotic tubulin structures have not been clarified. In this study, we used immunofluorescent staining of centromere protein B (CENP-B) with the formed micronuclei, and that of β- and γ-tubulin reflecting the structures of mitotic spindle and centrioles, respectively, in V79-hCYP1A2-hSULT1A1 cells. The results indicated that 1-MP induced micronuclei in V79-hCYP1A2-hSULT1A1 cells from 0.125 to 2 μM under a 24 h/0 h (exposure/recovery) regime, while in the parental V79-Mz cells micronuclei were induced by 1-MP only at concentrations ≥ 8 μM; in both cases, the micronuclei induced by 1-MP were predominantly CENP-B positive. Following 54 h of exposure, 1-MP induced mitotic spindle non-congression and centrosome amplification (multipolar mitosis) in V79-hCYP1A2-hSULT1A1 cells, and anaphase/telophase retardation, at concentrations ≥ 0.125 μM with concentration-dependence; while in V79-Mz cells it was inactive up to 8 μM. This study suggests that in mammalian cells proficient in activating enzymes 1-MP may induce chromosome loss and mitotic disturbance, probably by interfering with the mitotic spindle and centrioles.

Sequence Analysis and FISH Mapping of Four Satellite DNA Families among Cervidae.

Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.

The CENP-B box, a nucleotide motif involved in centromere formation, has multiple origins in New World monkeys.

Centromere protein B (CENP-B), a protein participating in centromere formation, binds to centromere satellite DNA by recognizing a 17-bp motif called the CENP-B box. This motif is found in hominids (humans and great apes) at an identical location in repeat units of their centromere satellite DNA. We have recently reported that the CENP-B box exists at diverse locations in three New World monkey species (marmoset, squirrel monkey and tamarin). However, the evolutionary origin of the CENP-B box in these species was not determined. It could have been present in a common ancestor, or emerged multiple times in different lineages. Here we present results of a phylogenetic analysis of centromere satellite DNA that support the multiple emergence hypothesis. Repeat units almost invariably formed monophyletic groups in each species and the CENP-B box location was unique for each species. The CENP-B box is not essential for the immediate survival of its host organism. On the other hand, it is known to be required for de novo centromere assembly. Our results suggest that the CENP-B box confers a long-term selective advantage. For example, it may play a pivotal role when a centromere is accidentally lost or impaired.

Reproducibility of fluid-phase measurements in PBS-treated sputum supernatant of healthy and stable COPD subjects.

PurposeThe purpose of this study was to investigate the reproducibility of fluid-phase measurements in PBS-treated sputum supernatant, processed using the two-step method, of healthy and stable COPD individuals.MethodsNine healthy subjects and 23 stable COPD patients provided sputum twice within 6 days. A two-step sputum processing method was used to obtain PBS-treated supernatant and sputum cells. Soluble protein markers and IgG and IgM autoantibody profiles in PBS supernatant were analyzed using customized microarrays. Repeatability of measurements was assessed by paired-sample testing and an intraclass correlation coefficient, then graphically reported by Bland–Altman plot.ResultsThere was no significant difference between the repeated detection of 8/10 types of soluble protein markers, all 13 types of IgG autoantibodies, and 12/13 types of corresponding IgM autoantibodies in PBS supernatant. The repeatability of measurements in PBS supernatant was substantial to very good for interleukin 6 (IL6), IL8, IL13, IL10, IL33, vascular endothelial growth factor, soluble receptor for advanced glycation end-products, and tumor necrosis factor-α; for IgG autoantibodies against aggrecan, centromere protein B (CENP-B), collagen II, collagen IV, cytochrome C, elastin, heat shock protein 47 (HSP47), HSP70, and La/Sjögren syndrome type B antigen; for IgM autoantibodies against CENP-B, collagen I, collagen II, collagen IV, cytokeratin 18, and HSP70; and for sputum neutrophils, macrophages and eosinophils count. Bland–Altman plots suggested good consistency within repeated measurements. Stable COPD patients differed from healthy subjects in the proportion of neutrophils and eosinophils; relative fluorescence intensity of anti-cytochrome C IgG, anti-aggrecan IgM, and anti-cytochrome C IgM. There was a significant positive correlation for stable COPD patients between sputum anti-collagen II IgG and post-bronchodilator FEV1%.ConclusionWe confirmed fluid-phase measurements in PBS-treated sputum supernatant by high-throughput techniques with good repeatability. We demonstrated the presence of IgG and IgM autoantibodies to multiple antigens in the airways of COPD patients.

Disease-related autoantibody profile in patients with systemic sclerosis

Background: Autoantibodies (autoAbs) help in diagnosis and predicting clinical phenotypes in systemic sclerosis (SSc).Aim of the study: To determine the clinical utility of 13 SSc-related autoAbs in SSc patients.Material and methods: A total of 131 consecutive patients with SSc (111 female, mean age 58.1 ± 14 years; 49 with diffused cutaneous SSc [dcSSc] and 82 with limited cutaneous SSc [lcSSc]) were analysed by a multiplex line immunoassay (Euroimmun) for autoantibodies (autoAbs) against 13 SSc-related antigens. A total of 22 patients with primary Raynaud phenomenon (RP), and 22 healthy controls were also analysed.Results: ANA by indirect immunofluorescence was present in 128 (97.7%) patients with SSc. Excluding anti-Ro52, 113 (89.3%) SSc patients were positive for at least one autoAb: anti-Topoisomerase I (anti-Topo) I abs in 54 (41.2%), anti-centromere proteins (anti-CENP) in 37 (28.2%, all reactive with centromere protein-A (CENPA) and centromere protein B (CENPB)), anti-RNA polymerase III(RP11) in 19 (14.5%), anti-RNA polymerase III(RP155) in 13 (9.9%), anti-fibrillarin in 4 (3.1%), anti-Ku in 6 (4.6%), anti-nucleolus-organizing region (anti-NOR90) in 8 (6.1%), anti-PM-Scl100 in 2 (1.5%), and anti-PM-Scl75 in 4 (3.1%). There was no immunoreactivity for Th/To or platelet-derived growth factor receptor (PDGFR). Overall, 102 (77.9%) SSc patients had autoAbs against Topo I, CENPA or CENPB, RP11 or RP155. Anti-Topo I abs were strongly associated with dcSSc, interstitial lung disease (ILD) (p < .001), pulmonary hypertension (PH) (p = .019) and ILD-PH (p = .003). Anti-CENPB abs were associated with lcSSc, and negatively associated with ILD. Anti-RP11 and anti-NOR90 abs were associated with male gender, and anti-NOR90 associated with ILD.Conclusions: Anti-Topo I, anti-CENP, and anti-RNA pol III are the most prevalent autoAbs in SSc. Anti-Topo I and anti-NOR90 abs are associated with ILD and/or PAH.

Mutational spectrum of CENP-B box and α-satellite DNA on chromosome 21 in Down syndrome children

The centromere is responsible for the correct inheritance of eukaryotic chromosomes during cell division. Centromere protein B (CENP‑B) and its 17 base pair binding site (CENP‑B box), which appears at regular intervals in centromeric α-satellite DNA (α-satDNA), are important for the assembly of the centromere components. Therefore, it is conceivable that CENP-B box mutations may induce errors in cell division. However, the association between the deoxynucleotide alterations of the CENP‑B box and the extra chromosome 21 (Chr21) present in patients with Down syndrome (DS) remains to be elucidated. The mutational spectrum of the α‑satDNA, including 4 functional CENP‑B boxes in Chr21 from 127 DS and 100 healthy children were analyzed by direct sequencing. The denovo occurrences of mutations within CENP‑B boxes in patients with DS were excluded. The prevalence of 6 novel mutations (g.661delC, g.1035_1036insA, g.1076_1077insC, g.670T>G, g.1239A>T, g.1343T>C) and 3 single nucleotide polymorphisms (g.727C/T, g.863A/C, g.1264C/G) were not significantly different between DS and controls (P>0.05). However, g.525C/G (P=0.01), g.601T/C (P=0.00000002), g.1279A/G (P=0.002), g.1294C/T (P=0.0006) and g.1302G/T (P=0.004) were significantly associated with the prevalence of DS (P<0.05). The results indicated that CENP‑B boxes are highly conserved in DS patients and may not be responsible for Chr21 nondisjunction events. However, α‑satDNA in Chr21 is variable and deoxynucleotide deletions, mutations and polymorphisms may act as potential molecular diagnostic markers of DS.

CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey.

Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought.

HTLV-1 bZIP factor suppresses the centromere protein B (CENP-B)-mediated trimethylation of histone H3K9 through the abrogation of DNA-binding ability of CENP-B.

Human T-cell leukaemia virus type-1 (HTLV-1) infection causes adult T-cell leukaemia (ATL). The viral protein HTLV-1 bZIP factor (HBZ) is constitutively expressed in ATL cells, suggesting that HBZ plays a major role in the pathogenesis of HTLV-1-associated disease. Here, we identified centromere protein B (CENP-B) as a novel interacting partner of HBZ. HBZ and CENP-B associate with their central regions in cells. Furthermore, overexpression of HBZ abrogated the DNA-binding activity of CENP-B to the α-satellite DNA region containing the CENP-B box motif, which in turn inhibited the CENP-B-mediated trimethylation of histone H3K9 in T-cells.

Classification of DNA minor and major grooves binding proteins according to the NLSs by data analysis methods.

High-mobility group proteins are a superfamily of DNA-binding proteins that bind to the DNA minor groove and bend it, whereas most of the transcription factors such as centromere protein B (CENP-B), octamer (Oct)-1, growth factor independence 1 (Gfi-1), and WRKY bind to the major groove of DNA. Classification of proteins using their DNA-binding features is the aim of this study. Nuclear localization signals play more important roles in entering DNA-binding proteins to nucleus and doing their functions; therefore, they have been considered as a feature which is important for DNA-binding manner in proteins. Nuclear localization signals (NLSs) were predicted by two prediction web servers, and then, their sequence ordered features were extracted by Chou's pseudo amino acid composition (PseAAC) and ProtParam. Multilayer perceptron was used as an artificial neural network for analyzing the features by calculating the correlation coefficient and 30-fold cross-validation. Another used data-analyzing program was principal component analysis of the Minitab software. By calculating the eigenvalues and considering five principal components, the sequence length of NLSs was known as the best feature for classifying DNA-binding proteins. Minimum mean squared error (MSE) (0.1098) and the highest R (2) (0.963) mean that there is a significant difference between the NLS length of the DNA major groove and minor groove binder proteins. Results showed that it is possible to classify DNA major groove and minor groove binder proteins by their NLS sequences as a feature.

Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system

Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture.

Identification of Novel α-N-Methylation of CENP-B That Regulates Its Binding to the Centromeric DNA

The eukaryotic centromere is an essential chromatin region required for accurate segregation of sister chromatids during cell division. Centromere protein B (CENP-B) is a highly conserved protein which can bind to the 17-bp CENP-B box on the centromeric DNA. In this study, we found that CENP-B could be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-B's binding to CENP-B box in cells. Our study also expands the function of protein α-N-methylation that has been known for decades and whose function remains largely unexplored.

AB0495 Audiovestibular involvement in systemic sclerosis: our exprerince

BackgroundHearing and balance disorders may occur in systemic sclerosis (SSc) as a result of the immune-mediated inner ear damage, the etiology being vasculitis and fibrosis. Objective of our study was...