KRAB-associated Protein Research Articles

CRISPR screening uncovers nucleolar RPL22 as a heterochromatin destabilizer and senescence driver.

Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.

Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Destabilizing heterochromatin by APOE mediates senescence.

Apolipoprotein E (APOE) is a component of lipoprotein particles that function in the homeostasis of cholesterol and other lipids. Although APOE is genetically associated with human longevity and Alzheimer's disease, its mechanistic role in aging is largely unknown. Here, we used human genetic, stress-induced and physiological cellular aging models to explore APOE-driven processes in stem cell homeostasis and aging. We report that in aged human mesenchymal progenitor cells (MPCs), APOE accumulation is a driver for cellular senescence. By contrast, CRISPR-Cas9-mediated deletion of APOE endows human MPCs with resistance to cellular senescence. Mechanistically, we discovered that APOE functions as a destabilizer for heterochromatin. Specifically, increased APOE leads to the degradation of nuclear lamina proteins and a heterochromatin-associated protein KRAB-associated protein 1 via the autophagy-lysosomal pathway, thereby disrupting heterochromatin and causing senescence. Altogether, our findings uncover a role of APOE as an epigenetic mediator of senescence and provide potential targets to ameliorate aging-related diseases.

Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells.

Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.

RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I (encoded by Ddx58) and melanoma differentiation-associated gene 5 (MDA5) (encoded by Ifih1), are crucial for initiating antiviral responses. Endogenous retroviral elements (ERVs) are transposable elements derived from exogenous retroviruses that are integrated into the genome. KRAB-associated protein 1 (KAP1) is a key epigenetic suppressor of ERVs that protects cells from detrimental genome instability. Increased ERV transcripts are sensed by RLRs and trigger innate immune signaling. However, whether KAP1 directly controls RLRs activity remains unclear. In this study, we show that KAP1 attenuates RNA viral infection-induced type I IFNs and facilitates viral replication by inhibiting RIG-I/MDA5 expression in primary peritoneal macrophages (PMs) of C57BL/6J mice. Kap1 deficiency increases IFN-β expression and inhibits vesicular stomatitis virus replication in C57BL/6J mice in vivo. Mechanistically, KAP1 binds to the promoter regions of Ddx58 and Ifih1 and promotes the establishment of repressive histone marks in primary PMs of C57BL/6J mice. Concordantly, KAP1 suppresses the expression of RIG-I and MDA5 at the transcriptional level in primary PMs of C57BL/6J mice. Our results establish that KAP1 epigenetically suppresses host antiviral responses by directly targeting RIG-1 and MDA5, thus facilitating the immune escape of RNA viruses.

Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

KAP1 targets actively transcribed genomic loci to exert pleomorphic effects on RNA polymerase II activity.

KAP1 (KRAB-associated protein 1) is best known as a co-repressor responsible for inducing heterochromatin formation, notably at transposable elements. However, it has also been observed to bind the transcription start site of actively expressed genes. To address this paradox, we characterized the protein interactome of KAP1 in the human K562 erythro-leukaemia cell line. We found that the regulator can associate with a wide range of nucleic acid binding proteins, nucleosome remodellers, chromatin modifiers and other transcription modulators. We further determined that KAP1 is recruited at actively transcribed polymerase II promoters, where its depletion resulted in pleomorphic effects, whether expression of these genes was normally constitutive or inducible, consistent with the breadth of possible KAP1 interactors.This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage.

Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

ZFP30 promotes adipogenesis through the KAP1-mediated activation of a retrotransposon-derived Pparg2 enhancer

Krüppel-associated box zinc finger proteins (KZFPs) constitute the largest family of mammalian transcription factors, but most remain completely uncharacterized. While initially proposed to primarily repress transposable elements, recent reports have revealed that KFZPs contribute to a wide variety of other biological processes. Using murine and human in vitro and in vivo models, we demonstrate here that one poorly studied KZFP, ZFP30, promotes adipogenesis by directly targeting and activating a retrotransposon-derived Pparg2 enhancer. Through mechanistic studies, we further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context. Our findings provide an understanding of both adipogenic and KZFP-KAP1 complex-mediated gene regulation, showing that the KZFP-KAP1 axis can also function in a non-repressive manner.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical roleof CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes

Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.

High expression of hnRNPAB/Kap1 together promote poor prognosis in HCC

Objective: To further understand the interaction protein spectrum of heterogeneous ribonucleoprotein AB (hnRNP AB), and to investigate their clinical significance in hepatocellular carcinoma (HCC). Methods: We carried out mass spectrometry to reveal the specific peptides of KRAB-associated protein 1 (Kap1) and hnRNPAB, and verified their interaction by immunocoprecipitation and western blotting. Expression of hnRNPAB/Kap1 proteins were detected by immunohistochemical staining in the tissue microarrays. Categorical data were analyzed by the chi square test or Fisher exact test; enumeration data between groups were compared using Student t-test or Wilcocon signed rank test; the cumulative recurrence and survival rates were evaluated using the Kaplan-Meier method and the differences were assessed using the log-rank test. Results: We identified Kap1 as a molecular partner for hnRNPAB in HCCLM3 cells and HepG2 cells as well. We found that the 5-year survival rate of the Kap1high patients was significantly lower than the survival rate of those of the Kap1low group (36% vs 59% , HR = 1.67, P < 0.001). Similarly, Kap1high HCC patients had the poorest prognosis at 5-years, with higher cumulative recurrence rate than Kap1low patients (72% vs 54%, HR = 1.66, P = 0.001). Univariate and Multivariate analyses revealed that hnRNPAB /Kap1 alone (HR = 1.35 /1.28, P = 0.001) or in combination with Kap1 (HR =1.24 /1.27, P < 0.05) were independent prognostic indicators for overall survival and time to recurrence. Conclusion: In HCC cells, hnRNPAB and Kap1 form protein complexes. The expression levels of hnRNPAB alone or in combination with Kap1 in HCC patients are important because they provide not only a predictor for HCC prognosis but also a therapeutic target for future studies.

Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription.

Anti-Inflammatory Activity of Sanghuangporus sanghuang Mycelium.

Acute lung injury (ALI) is characterized by inflammation of the lung tissue and oxidative injury caused by excessive accumulation of reactive oxygen species. Studies have suggested that anti-inflammatory or antioxidant agents could be used for the treatment of ALI with a good outcome. Therefore, our study aimed to test whether the mycelium extract of Sanghuangporus sanghuang (SS-1), believed to exhibit antioxidant and anti-inflammatory properties, could be used against the excessive inflammatory response associated with lipopolysaccharides (LPS)-induced ALI in mice and to investigate its possible mechanism of action. The experimental results showed that the administration of SS-1 could inhibit LPS-induced inflammation. SS-1 could reduce the number of inflammatory cells, inhibit myeloperoxidase (MPO) activity, regulate the TLR4/PI3K/Akt/mTOR pathway and the signal transduction of NF-κB and MAPK pathways in the lung tissue, and inhibit high mobility group box-1 protein 1 (HNGB1) activity in BALF. In addition, SS-1 could affect the synthesis of antioxidant enzymes Heme oxygenase 1 (HO-1) and Thioredoxin-1 (Trx-1) in the lung tissue and regulate signal transduction in the KRAB-associated protein-1 (KAP1)/nuclear factor erythroid-2-related factor Nrf2/Kelch Like ECH associated Protein 1 (Keap1) pathway. Histological results showed that administration of SS-1 prior to induction could inhibit the large-scale LPS-induced neutrophil infiltration of the lung tissue. Therefore, based on all experimental results, we propose that SS-1 exhibits a protective effect against LPS-induced ALI in mice. The mycelium of S. sanghuang can potentially be used for the treatment or prevention of inflammation-related diseases.

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

γ-H2AX/53BP1/pKAP-1 foci and their linear tracks induced by in vitro exposure to radon and its progeny in human peripheral blood lymphocytes.

The biodosimetric information is critical for evaluating the human health hazards caused by radon and its progeny. Here, we demonstrated that the formation of phosphorylated histone variant H2AX (γ-H2AX), p53-binding protein 1 (53BP1) and phosphorylated KRAB-associated protein 1 (pKAP-1) foci and their linear tracks in human peripheral blood lymphocytes (HPBLs) in vitro exposed to radon and its progeny were dependent on the cumulative absorbed dose of radon exposure but was unrelated to the concentration of radon. Among them, γ-H2AX foci and its linear tracks were the most sensitive indicators with the lowest estimable cumulative absorbed dose of 1.74 mGy from their linear dose-response curves and sustained for 12 h after termination of radon exposure. In addition, three types of foci showed an overdispersed non-Poisson distribution in HPBLs. The ratios of pKAP-1/γ-H2AX foci co-localization, 53BP1/γ-H2AX foci co-localization and 53BP1/pKAP-1 foci co-localization were significantly increased in HPBLs exposed to radon while they were unrelated to the cumulative dose of radon exposure, suggesting that γ-H2AX, pKAP-1 and 53BP1 play an important role in the repair of heterochromatic double-strand breaks. Altogether, our findings provide an experimental basis for estimating the biological dose of internal α-particle irradiation from radon and its progeny exposure in humans.

ATM function and its relationship with ATM gene mutations in chronic lymphocytic leukemia with the recurrent deletion (11q22.3-23.2).

Approximately 10–20% of chronic lymphocytic leukemia (CLL) patients exhibit del(11q22–23) before treatment, this cohort increases to over 40% upon progression following chemoimmunotherapy. The coding sequence of the DNA damage response gene, ataxia-telangiectasia-mutated (ATM), is contained in this deletion. The residual ATM allele is frequently mutated, suggesting a relationship between gene function and clinical response. To investigate this possibility, we sought to develop and validate an assay for the function of ATM protein in these patients. SMC1 (structural maintenance of chromosomes 1) and KAP1 (KRAB-associated protein 1) were found to be unique substrates of ATM kinase by immunoblot detection following ionizing radiation. Using a pool of eight fluorescence in situ hybridization-negative CLL samples as a standard, the phosphorylation of SMC1 and KAP1 from 46 del (11q22–23) samples was analyzed using normal mixture model-based clustering. This identified 13 samples (28%) that were deficient in ATM function. Targeted sequencing of the ATM gene of these samples, with reference to genomic DNA, revealed 12 somatic mutations and 15 germline mutations in these samples. No strong correlation was observed between ATM mutation and function. Therefore, mutation status may not be taken as an indicator of ATM function. Rather, a direct assay of the kinase activity should be used in the development of therapies.

Functional and Structural Features of Zinc Finger Protein 809

Zinc finger protein 809 (ZFP809) is a member of the kruppel-associated boxcontaining ZFP (KRAB-ZFP) family. It supresses the expression of Moloney murine leukemia virus (MoMLV) via sequence-specific binding to the primer binding site (PBS) located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at the LTR, such as repressive histone modifications and de novo DNA methylation. Recent studies have demonstrated the features of diverse functional domains of ZFP809, which has a KRAB domain and seven zinc fingers. We previously demonstrated that the KRAB domain is essential for nuclear localization, gene silencing, and binding to the MLV-PBS in conjunction with the accessory roles of a subset of zinc fingers. Individual zinc fingers are known to contribute to binding to the MLV-PBS and stable protein expression. Furthermore, the mechanisms underlying the high expression of ZFP809 in immature cells have yet to be fully elucidated. Recent studies have indicated that ZFP809 is stably expressed in immature cells, such as embryonic stem cells (ESCs), as ESCs have higher expression of KRAB-associated protein 1 (KAP1) than mature mouse embryonic fibroblast cells. Here we discuss the ZFP809/KAP1 complex, functional domains of ZFP809, and expression pattern of ZFP809 in immature cells.