Use of high throughput sequencing to observe genome dynamics at a single cell level.

With the development of high throughput sequencing technology, it becomes possible to directly analyze mutation distribution in a genome-wide fashion, dissociating mutation rate measurements from the traditional underlying assumptions. Here, we sequenced several genomes of Escherichia coli from colonies obtained after chemical mutagenesis and observed a strikingly nonrandom distribution of the induced mutations. These include long stretches of exclusively G to A or C to T transitions along the genome and orders of magnitude intra- and intergenomic differences in mutation density. Whereas most of these observations can be explained by the known features of enzymatic processes, the others could reflect stochasticity in the molecular processes at the single-cell level. Our results demonstrate how analysis of the molecular records left in the genomes of the descendants of an individual mutagenized cell allows for genome-scale observations of fixation and segregation of mutations, as well as recombination events, in the single genome of their progenitor.

The p400 complex is an essential E1A transformation target.

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.

Mammalian histone acetyltransferases and their complexes.

One of the key questions in the current molecular genetics of eukaryotes is how genetic information is retrieved from tightly packed chromatin. Acetylation of core histone N-termini is implicated in the regulation of chromatin function, and I summarize what is known about the mammalian enzymes that promote this posttranslational histone modification. Chromatin is important in gene expression not only because of the accessibility problem that it poses for the transcriptional machinery but also with regard to the phenomenon of chromatin memory, i.e. the ability of alternative chromatin states to be maintained through many cell divisions. This phenomenon is believed to be central to epigenetic inheritance, an important concept in developmental biology, which is also emerging as a contributing factor in cancer and other health disorders. Analyses of the composition of large multiprotein acetyltransferase complexes suggest their role in the mechanisms of epigenetic inheritance. The review will discuss some models pertinent to this function of histone acetyltransferases.

Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcomas, body cavity-based lymphomas, and some forms of Castleman's disease. The K9 open reading frame of KSHV encodes a viral interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction and as an oncogene to induce cell growth transformation. We demonstrate that KSHV vIRF directly interacts with cellular transcriptional coactivator p300 and displaces p300/CBP-associated factor from p300 complexes. This interaction inhibits the histone acetyltransferase activity of p300, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure. As a consequence, vIRF expression markedly alters cellular cytokine expression, which is regulated by acetylation of nucleosomal histones. These results demonstrate that KSHV vIRF interacts with and inhibits the p300 transcriptional coactivator to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression. These studies also illustrate how a cellular gene captured by a herpesvirus has evolved several functions that suit the needs of the virus.

Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Properties of the glucocorticoid modulatory element binding proteins GMEB-1 and -2: potential new modifiers of glucocorticoid receptor transactivation and members of the family of KDWK proteins.

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at -3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation.

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [(14)C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.

An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression.

In order to study interferon regulatory factor (IRF) family mediation of cell growth regulation, we established U937 cell lines stably transfected with a truncated form of IRF-2 lacking the transcriptional repressor domain. The truncated IRF-2 contained the DNA binding domain (DBD) and bound the ISRE. Phenotypically, the IRF-2 DBD transfectants exhibited reduced cell growth, altered morphology and increased cell death. Consistent with alterations in growth characteristics, the IRF-2 DBD transfectants constitutively expressed higher levels of the cyclin dependent kinase inhibitor p21WAF1/Cip1 than did control clones. The level of p21WAF1/Cip1 expression was positively correlated with the level of DBD expressed, as well as with the level of growth inhibition in these clones. DBD expression also correlated with expression of other members of the growth regulatory complex, cyclin dependent kinase 2 and cyclin A, but not proliferating cell nuclear antigen. These results imply active repression by IRF-2 to keep p21WAF1/Cip1 transcriptionally silent.

Mammalian histone acetyltransferase complexes.

Over the last decade, great progress has been made in elucidating how the human genome operates in the chromatin context. This paper describes our work on two human acetyltransferases, PCAF and TIP60, and their interaction partners. This study provides new clues on the function of these enzymes. In a striking parallel with the general transcription factor TFIID, PCAF complex contains proteins that have histone-like domains. We speculate that these subunits can presumably form a nucleosome-like structure on DNA, which would allow PCAF to contribute to the maintenance of an active state of chromatin. On the other hand, TIP60 complex contains two eukaryotic homologs of bacterial RuvB helicase/ATPse, involved in recombination and repair. Accordingly, expression of a dominant negative mutant of TIP60 in living cells interferes with their ability to repair DNA damage, which points out, for the first time, a role for a histone acetyltransferase in a process other than transcription. We also have evidence implicating TIP60 in the apoptotic response to DNA damage.

Mammalian histone acetyltransferase complexes

Over the last decade, great progress has been made in elucidating how the human genome operates in the chromatin context. This paper describes our work on two human acetyltransferases, PCAF and TIP60, and their interaction partners. This study provides new clues on the function of these enzymes. In a striking parallel with the general transcription factor TFIID, PCAF complex contains proteins that have histone-like domains. We speculate that these subunits can presumably form a nucleosome-like structure on DNA, which would allow PCAF to contribute to the maintenance of an active state of chromatin. On the other hand, TIP60 complex contains two eukaryotic homologs of bacterial RuvB helicase/ATPse, involved in recombination and repair. Accordingly, expression of a dominant negative mutant of TIP60 in living cells interferes with their ability to repair DNA damage, which points out, for the first time, a role for a histone acetyltransferase in a process other than transcription. We also have evidence implicating TIP60 in the apoptotic response to DNA damage.

Mammalian histone acetyltransferase complexes.

Over the last decade, great progress has been made in elucidating how the human genome operates in the chromatin context. This paper describes our work on two human acetyltransferases, PCAF and TIP60, and their interaction partners. This study provides new clues on the function of these enzymes. In a striking parallel with the general transcription factor TFIID, PCAF complex contains proteins that have histone-like domains. We speculate that these subunits can presumably form a nucleosome-like structure on DNA, which would allow PCAF to contribute to the maintenance of an active state of chromatin. On the other hand, TIP60 complex contains two eukaryotic homologs of bacterial RuvB helicase/ATPse, involved in recombination and repair. Accordingly, expression of a dominant negative mutant of TIP60 in living cells interferes with their ability to repair DNA damage, which points out, for the first time, a role for a histone acetyltransferase in a process other than transcription. We also have evidence implicating TIP60 in the apoptotic response to DNA damage.

PCAF interacts with tax and stimulates tax transactivation in a histone acetyltransferase-independent manner.

Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity.

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A.

Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein.

Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program.

p300/CBP and PCAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation. They are both required for MyoD activity and muscle differentiation. Nevertheless, their roles must be different since the acetyltransferase activity of PCAF but not of p300 is involved in controlling myogenic transcription and differentiation. Here, we provide a molecular explanation of this phenomenon and report that MyoD is directly acetylated by PCAF at evolutionarily conserved lysines. Acetylated MyoD displays an increased affinity for its DNA target. Importantly, conservative substitutions of acetylated lysines with nonacetylatable arginines impair the ability of MyoD to stimulate transcription and to induce muscle conversion indicating that acetylation of MyoD is functionally critical.

Regulation of activity of the transcription factor GATA-1 by acetylation.

Modification of histones, DNA-binding proteins found in chromatin, by addition of acetyl groups occurs to a greater degree when the histones are associated with transcriptionally active DNA. A breakthrough in understanding how this acetylation is mediated was the discovery that various transcriptional co-activator proteins have intrinsic histone acetyltransferase activity (for example, Gcn5p, PCAF, TAF(II)250 and p300/CBP. These acetyltransferases also modify certain transcription factors (TFIIEbeta, TFIIF, EKLF and p53). GATA-1 is an important transcription factor in the haematopoietic lineage and is essential for terminal differentiation of erythrocytes and megakaryocytes. It is associated in vivo with the acetyltransferase p300/CBP. Here we report that GATA-1 is acetylated in vitro by p300. This significantly increases the amount of GATA-1 bound to DNA and alters the mobility of GATA-1-DNA complexes, suggestive of a conformational change in GATA-1. GATA-1 is also acetylated in vivo and acetylation directly stimulates GATA-1-dependent transcription. Mutagenesis of important acetylated residues shows that there is a relationship between the acetylation and in vivo function of GATA-1. We propose that acetylation of transcription factors can alter interactions between these factors and DNA and among different transcription factors, and is an integral part of transcription and differentiation processes.

Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo.

We identify Xenopus NF-Y as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. We clone Xenopus NF-YA, NF-YB and NF-YC and establish that NF-Y is the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. We suggest two steps in chromatin modification at the Xenopus hsp70 promoter: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity.

Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat.

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

A human RNA polymerase II complex containing factors that modify chromatin structure.

We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.

Histone-like TAFs within the PCAF histone acetylase complex.

PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.