Recurrent chromosomal translocations in sarcomas create a megacomplex that mislocalizes NuA4/TIP60 to Polycomb target loci.

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.

The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation.

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.

Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex.

In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins ARS305 and ARS607. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.

Histone chaperones: modulators of chromatin marks.

The many factors that control chromatin biology play key roles in essential nuclear functions like transcription, DNA damage response and repair, recombination, and replication and are critical for proper cell-cycle progression, stem cell renewal, differentiation, and development. These players belong to four broad classes: histone modifiers, chromatin remodelers, histone variants, and histone chaperones. A large number of studies have established the existence of an intricate functional crosstalk between the different factors, not only within a single class but also between different classes. In light of this, while many recent reviews have focused on structure and functions of histone chaperones, the current text highlights novel and striking links that have been established between these proteins and posttranslational modifications of histones and discusses the functional consequences of this crosstalk. These findings feed a current hot question of how cell memory may be maintained through epigenetic mechanisms involving histone chaperones.

HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail.

The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor.

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle.

A balancing act: interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans.

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1's role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity.

Functions of myst family histone acetyltransferases and their link to disease.

The MYST family of histone acetyltransferases is highly conserved in eukaryotes and is responsible for the majority of acetylation events. These enzymes are exclusively found in multisubunit protein complexes, which structure is also very well conserved. Recent studies have shed light on the precise functions of these HAT complexes. They play critical roles in gene-specific transcription regulation, DNA damage response and repair, as well as DNA replication. Such roles in basic nuclear functions suggest that alteration of these MYST HAT complexes could lead to malfunctioning cells, leading to cell death, uncontrolled growth and/or disease. Indeed, many of these enzymes and their associated factors have been implicated in several forms of cancers. This chapter summarizes the current knowledge on MYST HAT complexes, their functions and link to human diseases.

Interaction of the HPV E7 proteins with the pCAF acetyltransferase.

Most cervical carcinomas express the E6 and E7 proteins of a high-risk human papillomavirus (HPV). These proteins affect growth control by interfering with the functions of cell regulatory proteins, promoting oncogenic transformation. A key target of E7 is the tumor suppressor protein pRb, which directly interacts with E7. However, binding to additional cellular regulatory proteins is clearly required for oncogenesis, as mutants of E7 have been identified that bind to pRb, yet fail to transform efficiently. Here we demonstrate the interaction of the HPV 6, 16 and 18 E7 proteins with the pCAF acetyltransferase, which has been reported to function as a coactivator for a variety of transcription factors including p53. Mutation of a highly conserved leucine residue within the zinc finger region of HPV 16 E7 disrupts binding to pCAF and also impairs transformation and transcriptional activation. HPV 16 E7 interacts with the acetyltransferase domain of pCAF, and reduces its acetyltransferase activity in vitro. Our analysis of the interaction between the pCAF acetyltransferase and E7 provides new insight into the mechanisms by which the E7 oncoproteins can alter cellular gene expression and growth.

Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300.

Infection with high-risk human papillomaviruses (HPV) can lead to the development of cervical cancer. This process depends on the interaction of the virus-encoded oncoproteins, E6 and E7, with a variety of host regulatory proteins. As E7 shares both functional and structural similarities with the Adenovirus E1a (Ad E1a) protein, we were interested in investigating the possible interactions between E7 and the transcriptional coactivator p300, since it was originally identified as a target of Ad E1a. Using a variety of assays, we show that E7s from both high- and low-risk HPV types interact with p300. Mutational analysis of E7 maps the site of the interaction to a region spanning the pRb-binding domain and the CKII phosphorylation site. We also map the site of interaction on p300 largely to the CH1 domain. In addition, we demonstrate that the binding between 16E7 and p300 is direct, and can be detected in vivo by coimmunoprecipitation and mammalian two-hybrid assays. Finally, we show that E7 can abolish the p300-mediated E2 transactivation function, suggesting that complex formation between E7 and p300 may contribute to the regulation of E2 transcriptional activity.

New tools for the construction of replication-competent adenoviral vectors with altered E1A regulation.

We have designed new vectors for the construction of recombinant adenoviruses containing the early region 1A (E1A) gene under the transcriptional control of heterologous promoters. The normal E1A regulatory elements have been replaced by a convenient set of unique restriction enzyme sites, allowing for introduction of gene regulatory cassettes with relative ease. Subsequent rescue generates recombinant conditionally replicating adenovirus in which the transcription of E1A is under alternative control. This allows potentially cell-type specific expression of E1A, restricting efficient virus replication to chosen target cells. It is shown that in several viruses rescued using these constructs, E1A expression is regulated by the heterologous promoters in the expected manner. Specifically, a virus with E1A under the control of the human Cytomegalovirus Immediate Early promoter produced constitutively high levels of E1A. A second virus, with E1A expression regulated by the glucocorticoid-responsive Mouse Mammary Tumor Virus promoter produced E1A expression in a dose-dependent manner upon dexamethasone treatment. Efficient growth of this second virus also required the presence of dexamethasone.

Histone phosphorylation: a chromatin modification involved in diverse nuclear events.

Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes.

Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation.

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.

Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1.

The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here we show that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9-Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate Lys27 of histone H3 in vitro and in vivo. Laser microirradiation data show that PHF1 is transiently recruited to DNA double-strand breaks, and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at double-strand break sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as a cofactor in early DNA-damage response.

MOF and histone H4 acetylation at lysine 16 are critical for DNA damage response and double-strand break repair.

The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.

Radiochromic leuco dye micelle hydrogels: I. Initial investigation.

This investigation reports the use of surfactants and colorless leuco triarylmethane dyes to form a new class of radiochromic micelle hydrogels for three-dimensional (3D) water-equivalent dosimetry. Gelatin gel samples with several surfactants and leuco dyes were prepared and evaluated for optical transparency, dose sensitivity and diffusion rates. The addition of Triton X-100, a non-ionic surfactant, at levels exceeding the critical micelle concentration provides a transparent hydrogel in which the water insoluble leuco Malachite Green (LMG) can dissolve. During irradiation, the LMG dye precursor converts to Malachite Green (MG(+)). The most sensitive reported LMG gel formulation contains 0.3 mM LMG leuco dye, 16 mM trichloroacetic acid, 7 mM Triton X-100 and 4% w/w gelatin. A diffusion coefficient of 0.14 mm(2) h(-1) was determined for MG(+) in this gel by fitting the time-dependent degradation of the transmission profile after irradiating half of the sample. The diffusion rate was three times lower than the standard radiochromic ferrous xylenol-orange (FX) gel. The primary feature of this 3D hydrogel is that it introduces transparent, radiochromic, micelle hydrogels. The radiochromic response to dose is instantaneous and images are stable for several hours. A dosimetric characterization revealed that the dose response is reproducible to within 10% over five separate batches and independent of both energy and dose rate. Uniform pre-irradiation of samples to 5 Gy provided a subsequent near linear response to greater than 110 Gy. LMG gels when read with a fast optical CT scanner can provide full 3D dose distributions in less than 30 min post-irradiation. LMG micelle gels scanned with a 633 nm light source are a promising system for quantitative water- or tissue-equivalent 3D dose verification in the 5-100 Gy dose range. These gels are useful for the scanning of larger volume dosimeters (i.e. >15 cm diameter) since they are easily prepared with inexpensive ingredients, are transparent and have a low initial optical absorbance prior to irradiation. In this gel, only one colored molecular species is produced. This results in a linear relation between the concentration of the colored dye product and optical absorption independent of the sampled wavelength.

The E1A proteins of all six human adenovirus subgroups target the p300/CBP acetyltransferases and the SAGA transcriptional regulatory complex.

The N-terminal/conserved region 1 (CR1) portion of the human adenovirus (Ad) 5 E1A protein was previously shown to inhibit growth in the simple eukaryote Saccharomyces cerevisiae. We now demonstrate that the corresponding regions of the E1A proteins of Ad3,-4,-9,-12, and -40, which represent the remaining five Ad subgroups, also inhibit yeast growth. These results suggest that the E1A proteins of all six human Ad subgroups share a common cellular target(s) conserved in yeast. Growth inhibition induced by either full-length or the N-terminal/CR1 portion of Ad5 E1A was relieved by coexpression of the E1A binding portions of the mammalian p300, CBP, and pCAF acetyltransferases. Similarly, growth inhibition by the N-terminal/CR1 portions of the other Ad E1A proteins was suppressed by expression of the same regions of CBP or pCAF known to bind Ad5 E1A. The physical interaction of each of the different Ad E1A proteins with CBP, p300, and pCAF was confirmed in vitro. Furthermore, deletion of the gene encoding yGcn5, the yeast homolog of pCAF and a subunit of the SAGA transcriptional regulatory complex, restored growth in yeast expressing each of the different Ad E1A proteins. This indicates that the SAGA complex is a conserved target of all Ad E1A proteins. Our results demonstrate for the first time that the p300, CBP, and pCAF acetyltransferases are common targets for the E1A proteins of all six human Ad subgroups, highlighting the importance of these interactions for E1A function.

The adenovirus E1A protein targets the SAGA but not the ADA transcriptional regulatory complex through multiple independent domains.

Expression of the adenovirus E1A protein in the simple eukaryote Saccharomyces cerevisiae inhibits growth. We tested four regions of E1A that alter growth and transcription in mammalian cells for their effects in yeast when expressed as fusions to the Gal4p DNA binding domain. Expression of the N-terminal/conserved region (CR) 1 or CR3, but not of the CR2 or the C-terminal portion of E1A, inhibited yeast growth. Growth inhibition was relieved by deletion of the genes encoding the yGcn5p, Ngg1p, or Spt7p components of the SAGA transcriptional regulatory complex, but not the Ahc1p component of the related ADA complex, indicating that the N-terminal/CR1 and CR3 regions of E1A target the SAGA complex independently. Expression of the pCAF acetyltransferase, a mammalian homologue of yGcn5p, also suppressed growth inhibition by either portion of E1A. Furthermore, the N-terminal 29 residues and the CR3 portion of E1A interacted independently with yGcn5p and pCAF in vitro. Thus, two separate regions of E1A target the yGcn5p component of the SAGA transcriptional activation complex. A subregion of the N-terminal/CR1 fragment spanning residues 30-69 within CR1 also inhibited yeast growth in a SAGA-dependent fashion. However, this region did not interact with yGcn5p or pCAF, suggesting that it makes a third contact with another SAGA component. Our results provide a new model system to elucidate mechanisms by which E1A and the SAGA complex regulate transcription and growth.

Comparative sequence analysis of the largest E1A proteins of human and simian adenoviruses.

The early region 1A (E1A) gene is the first gene expressed after infection with adenovirus and has been most extensively characterized in human adenovirus type 5 (hAd5). The E1A proteins interact with numerous cellular regulatory proteins, influencing a variety of transcriptional and cell cycle events. For this reason, these multifunctional proteins have been useful as tools for dissecting pathways regulating cell growth and gene expression. Despite the large number of studies using hAd5 E1A, relatively little is known about the function of the E1A proteins of other adenoviruses. In 1985, a comparison of E1A sequences from three human and one simian adenovirus identified three regions with higher overall levels of sequence conservation designated conserved regions (CR) 1, 2, and 3. As expected, these regions are critical for a variety of E1A functions. Since that time, the sequences of several other human and simian adenovirus E1A proteins have been determined. Using these, and two additional sequences that we determined, we report here a detailed comparison of the sequences of 15 E1A proteins representing each of the six hAd subgroups and several simian adenoviruses. These analyses refine the positioning of CR1, 2, and 3; define a fourth CR located near the carboxyl terminus of E1A; and suggest several new functions for E1A.