Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2

In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.     

TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.     

Death-associated protein kinase 1 is an IRF3/7-interacting protein that is involved in the cellular antiviral immune response

Interferon regulatory factor （IRF） 7 has been demonstrated to be a master regulator of virus-induced type I interferon production （IFN）, and it plays a central role in the innate immune response against viruses. Here, we identified death-associated protein kinase 1 （DAPK1） as an IRF7-interacting protein by tandem affinity purification （TAP）. Viral infection induced DAPKI-IRF7 and DAPKI-IRF3 interactions and overexpression of DAPK1 enhanced virus-induced activation of the interferon-stimulated response element （ISRE） and IFN-p promoters and the expression of the IFNB1 gene. Knockdown of DAPK1 attenuated the induction of IFNB1 and RIG.lexpression triggered by viral infection or I FN-p, and they were enhanced by viral replication. In addition, viral infection or IFN-p treatment induced the expression of DAPK1. IFN-p treatment also activated DAPK1 by decreasing its phosphorylation level at serine 308. Interestingly, the involvement of DAPK1 in virus-induced signaling was independent of its kinase activity. Therefore, our study identified DAPK1 as an important regulator of the cellular antiviral response.     

Epe1 recruits BET family bromodomain protein Bdf2 to establish heterochromatin boundaries

Heterochromatin spreading leads to the silencing of genes within its path, and boundary elements have evolved to constrain such spreading. In fission yeast, heterochromatin at centromeres I and III is flanked by inverted repeats termed IRCs, which are required for proper boundary functions. However, the mechanisms by which IRCs prevent heterochromatin spreading are unknown. Here, we identified Bdf2, which is homologous to the mammalian bromodomain and extraterminal (BET) family double bromodomain proteins involved in diverse types of cancers, as a factor required for proper boundary function at IRCs. Bdf2 is enriched at IRCs through its interaction with the boundary protein Epe1. The bromodomains of Bdf2 recognize acetylated histone H4 tails and antagonize Sir2-mediated deacetylation of histone H4K16. Furthermore, abolishing H4K16 acetylation (H4K16ac) with an H4K16R mutation promotes heterochromatin spreading, and mimicking H4K16ac by an H4K16Q mutation blocks heterochromatin spreading at IRCs. Our results thus illustrate a mechanism of establishing chromosome boundaries at specific sites through the recruitment of a factor that protects euchromatic histone modifications. They also reveal a previously unappreciated function of H4K16ac in cooperation with H3K9 methylation to regulate heterochromatin spreading.     

Tpp1/Acd maintains genomic stability through a complex role in telomere protection

Telomeres serve to protect the ends of chromosomes, and failure to maintain telomeres can lead to dramatic genomic instability. Human TPP1 was identified as a protein which interacts with components of a telomere cap complex, but does not directly bind to telomeric DNA. While biochemical interactions indicate a function in telomere biology, much remains to be learned regarding the roles of TPP1 in vivo. We previously reported the positional cloning of the gene responsible for the adrenocortical dysplasia (acd) mouse phenotype, which revealed a mutation in the mouse homologue encoding TPP1. We find that cells from homozygous acd mice harbor chromosomes fused at telomere sequences, demonstrating a role in telomere protection in vivo. Surprisingly, our studies also reveal fusions and radial structures lacking internal telomere sequences, which are not anticipated from a simple deficiency in telomere protection. Employing spectral karyotyping and telomere FISH in a combined approach, we have uncovered a striking pattern; fusions with telomeric sequences involve nonhomologous chromosomes while those lacking telomeric sequences involve homologues. Together, these studies show that Tpp1/Acd plays a vital role in telomere protection, but likely has additional functions yet to be defined.     

A regulatory SNP of the BICD1 gene contributes to telomere length variation in humans

Telomeres are repetitive sequences of variable length at the ends of chromosomes involved in maintaining their integrity. Telomere dysfunction is associated with increased risk of cancer and other age-related diseases. Telomere length is an important determinant of telomere function and has a strong genetic basis. We previously carried out a genome-wide linkage analysis of mean leukocyte telomere length, and identified a 12 cm quantitative-trait locus affecting telomere length on human chromosome 12. In the present study we confirmed linkage to this locus in an extended sample (380 families, 520 sib-pairs, maximum LOD score 4.3). Fine-mapping identified a 51 kb region of association within intron 1 of the Bicaudal-D homolog 1 (BICD1, MIM 602204) gene. The strongest association (P = 1.9 x 10(-5)) was with SNP rs2630578 where the minor allele C (frequency 0.21) was associated with telomeres that were shorter by 604 (+/-204) base pairs, equivalent to approximately 15-20 years of age-related attrition in telomere length. Subjects carrying the C allele for rs2630778 had 44% lower BICD1 mRNA levels in their leukocytes compared with GG homozygotes (P = 0.004). BICD1 is involved in Golgi-to-endoplasmic reticulum vacuolar transport. Previous studies have implicated vacuolar genes in telomere length homeostasis in yeast. Our study indicates that BICD1 plays a similar role in humans.     

Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.     

Limited capacity of the nuclear matrix to bind telomere repeat binding factor TRF1 may restrict the proliferation of mortal human fibroblasts

The maintenance of telomere integrity is essential for prolonged cell proliferation, and failure in this mechanism is a most consistent manifestation of cellular senescence. In this study, we investigated the role of telomere repeat binding factor (TRF1) in the proliferation of human fibroblasts. TRF1 expression is upregulated in a large variety of immortal human cells and supports de novo telomere formation in a dose-dependent manner. These observations suggest that the suppression of TRF1 might limit telomere maintenance and thus the life span of mortal cells. However, primary fibroblasts ectopically overexpressing TRF1 were unable to avoid senescence. On the other hand, exogenously expressed TRF1 in primary fibroblasts neither supported de novo telomere formation nor bound to the nuclear matrix as tightly as observed in immortal cells that show upregulated TRF1 expression. We present evidence suggesting that mortal human cells lack specific ligand(s) that anchor TRF1 to the nuclear matrix and that this contributes to their limited lifespan.     

HIV-1 matrix protein: a mysterious regulator of the viral life cycle

Significant progress has been achieved in the last few years concerning the human immunodeficiency virus (HIV-1) life cycle, mostly in the fields of cellular receptors for the virus, virus assembly and budding of virus particles from the cell surface. Meanwhile, some aspects, such as postentry events, virus maturation and the regulatory role of individual viral proteins remain poorly defined. This review summarizes some recent findings concerning the role of Gag Pr55 and its proteolytic processing in the HIV-1 life cycle with particular emphasis on the functions of matrix protein p17 (MA), the protein which plays a key role in regulation of the early and late steps of viral morphogenesis. Based on our recent observations, the possibility is discussed that two subsets of MA exist, one cleaved from the Gag precursor in the host cell (cMA), and the other cleaved in the virions (vMA). It is suggested that two MA fractions possess diverse functions and are involved in different stages of virus morphogenesis as key regulators of the viral life cycle.     

Cyclin-dependent kinase 5, Munc18a and Munc18-interacting protein 1/X11alpha protein up-regulation in Alzheimer's disease

Besides formation of neurofibrillary tangles and neuron loss, the Alzheimer's disease brain is characterized by neuritic plaques consisting of beta-amyloid peptide deposits and impaired neurotransmission. The proteins Munc18a, Munc18-interacting protein 1 and Munc18-interacting protein 2 mediate exocytosis and decrease beta-amyloid peptide formation. Cyclin-dependent kinase 5 and its activator p35 disrupt Munc18a-syntaxin 1 binding, thereby promoting synaptic vesicle fusion during exocytosis. We investigated protein levels of the signaling pathway: p35, cyclin-dependent kinase 5, Munc18a, syntaxin 1A and 1B, Munc18-interacting protein 1 and Munc18-interacting protein 2 in Alzheimer's disease cortex and found that this pathway was up-regulated in the Alzheimer's disease parietal and occipital cortex. In the cortex of transgenic Tg2576 mice over-expressing human beta-amyloid precursor protein with the Swedish mutation known to lead to familial Alzheimer's disease, which have substantial levels of beta-amyloid peptide but lack neurofibrillary tangles and neuron loss, no alterations of protein levels were detected. These data suggest that the pathway is enhanced in dying or surviving neurons and might serve a protective role by compensating for decreased neurotransmission and decreasing beta-amyloid peptide levels early during the progression of Alzheimer's disease.     

Shelterin: the protein complex that shapes and safeguards human telomeres

Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.