Vasily Ogryzko

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.

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.

Immunoaffinity purification of mammalian protein complexes.

Publisher Summary This chapter aims to describe the generation of recombinant cells and the technique for purification of the complex. Identification of the interaction partners of a particular protein became a valuable way to gain insight in the physiological role of the protein. Many proteins appear to exist and function as stable multimeric protein complexes. For instance, the multimeric RNA polymerase II protein complex consists of 12 polypeptides. It transcribes DNA templates as a complex, whereas individual subunits have no such activity, thus making it impossible to predict “bona fide” functions of an RNA polymerase II from a single subunit. Likewise, if the protein of interest functions as a complex, it is crucial to purify it as a complex to determine what it does in the cell. The chapter also explores the most widely used system to identify intermolecular interactive partners of a particular protein of interest, the yeast two-hybrid system. The stably expressed exogenous proteins with epitope tags are integrated in the complexes when the complex is synthesized de novo. This chapter discusses such protein complexes purified rapidly by immunoprecipitation with antibodies against epitopes.

Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells.

Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair, and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net downregulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing.

YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity

Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3′-phosphoadenosine 5′-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

The Retinoblastoma Protein Binds the Promoter of the Survival Gene bcl-2 and Regulates Its Transcription in Epithelial Cells through Transcription Factor AP-2

ABSTRACT The retinoblastoma (RB) gene product has been shown to restrict cell proliferation, promote cell differentiation, and inhibit apoptosis. Loss of RB function can induce both p53-dependent apoptosis and p53-independent apoptosis; little is known about the mechanisms of RB-regulated p53-independent apoptosis. Here we show that RB specifically activates transcription of the survival gene bcl-2 in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated by the transcription factor AP-2. By monitoring protein-DNA interactions in living cells using formaldehyde cross-linking and chromatin immunoprecipitation, we show that endogenous RB and AP-2 both bind to the same bcl-2 promoter sequence. In addition, we demonstrate that RB and AP-2 also bind to the E-cadherin gene promoter in vivo, consistent with regulation of this promoter by both AP-2 and RB in epithelial cells. This study provides evidence that RB activates bcl-2 and E-cadherin by binding directly to the respective promoter sequences and not indirectly by repressing an inhibitor. This recruitment is mediated by a transcription factor, in this case AP-2. For the first time, our results suggest a direct molecular mechanism by which RB might inhibit apoptosis independently of p53. The results are discussed in a context where RB and Bcl-2 contribute under nonpathological conditions to the maintenance of cell viability in association with a differentiated phenotype, contributing to the tumor suppressor function of RB and playing important roles in normal development.

Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development

The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development.

Analysis of Human Histone H2AZ Deposition In Vivo Argues against Its Direct Role in Epigenetic Templating Mechanisms

ABSTRACT Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.

Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis

Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn– strain. A triple mutant (nrnA–, nrnB–, yhaM–) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5′-to-3′ exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.

Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.