Jordi Bernués

TFIID IS REQUIRED FOR IN VITRO TRANSCRIPTION OF THE HUMAN U6 GENE BY RNA POLYMERASE III

We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID‐containing fractions stimulate U6 transcription in reactions containing rate‐limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFIID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases.

The structure of a transcription activation subcomplex reveals how σ70 is recruited to PhoB promoters

PhoB is a two‐component response regulator that activates transcription by interacting with the σ70 subunit of the E. coli RNA polymerase in promoters in which the −35 σ70‐recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the σ4 domain of σ70 fused with the RNA polymerase β subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how σ4 recognizes the upstream pho box repeat. As with the −35 element, σ4 achieves this recognition through the N‐terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices α2 and α3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost −35 element contacts are compensated by new ones with the activator. In addition, σ4 is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

Common and unique transcription factor requirements of human U1 and U6 snRNA genes

The human U1 and U6 genes have similar basal promoter structures. A first analysis of the factor requirements for the transcription of a human U1 gene by RNA polymerase II in vitro has been undertaken, and these requirements compared with those of human U6 gene transcription by RNA polymerase III in the same extracts. Fractions containing PSE‐binding protein (PBP) are shown to be essential for transcription of both genes, and further evidence that PBP itself is required for U1 as well as U6 transcription is presented. On the other hand, the two genes have distinct requirements for TATA‐binding protein (TBP). On the basis of chromatographic and functional properties, the TBP, or TBP complex, required for U1 transcription appears to differ from previously described complexes required for RNA polymerase I, II or III transcription. The different TBP requirements of the U1 and U6 promoters are reflected by specific association with either TFIIB or TFIIIB respectively, thus providing a basis for differential RNA polymerase selection.

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

Drosophila melanogaster linker histone dH1 is required for transposon silencing and to preserve genome integrity

Histone H1 is an intrinsic component of chromatin, whose important contribution to chromatin structure is well-established in vitro. Little is known, however, about its functional roles in vivo. Here, we have addressed this question in Drosophila, a model system offering many advantages since it contains a single dH1 variant. For this purpose, RNAi was used to efficiently deplete dH1 in flies. Expression-profiling shows that dH1 depletion affects expression of a relatively small number of genes in a regional manner. Furthermore, depletion up-regulates inactive genes, preferentially those located in heterochromatin, while active euchromatic genes are down-regulated, suggesting that the contribution of dH1 to transcription regulation is mainly structural, organizing chromatin for proper gene-expression regulation. Up-regulated genes are remarkably enriched in transposons. In particular, R1/R2 retrotransposons, which specifically integrate in the rDNA locus, are strongly up-regulated. Actually, depletion increases expression of transposon-inserted rDNA copies, resulting in synthesis of aberrant rRNAs and enlarged nucleolus. Concomitantly, dH1-depleted cells accumulate extra-chromosomal rDNA, show increased γH2Av content, stop proliferation and activate apoptosis, indicating that depletion causes genome instability and affects proliferation. Finally, the contributions to maintenance of genome integrity and cell proliferation appear conserved in human hH1s, as their expression rescues proliferation of dH1-depleted cells.

Proximal sequence element factor binding and species specificity in vertebrate U6 snRNA promoters

The Xenopus tropicalis U6 gene is very poorly transcribed both when introduced into human cells by transfection, and in human cell-free extracts. By analysis of hybrid promoters constructed from human and Xenopus sequences in various combinations, we show that species specificity is mediated by the proximal sequence elements (PSEs) of the promoters. We demonstrate the PSE-dependence of U6 transcription in a fractionated extract of HeLa cells. One of the fractions required for transcription contains an activity designated PSE-binding protein (PBP), previously shown to bind to the PSE of the mouse U6 gene. Binding of PBP to various wild-type and hybrid U6 PSE sequences correlates with their activity in transcription in HeLa cell extracts. This provides strong evidence that PBP is the PSE-binding factor involved in U6 transcription. In addition, it suggests that the differential affinities of the promoters for PBP is responsible for the observed species specificity. The divergence between U snRNA promoters in different species contrasts with the relatively strong conservation of other families of RNA polymerase II and III transcribed gene promoters. Possible mechanisms by which this diversity could be generated are discussed.

Activation properties of GAGA transcription factor

GAGA is a Drosophila transcription factor that has been involved in many nuclear activities. We present evidence that GAGA factor enhances transcription by stabilizing pre-initiation complex (PIC) and promoting reinitiation. Formation of PIC prior to GAGA addition prevents activation suggesting that GAGA is required early in the formation of activated complexes. GAGA stimulation of transcription can be attributed in part to a stabilization of PIC. All these properties depend on the GAGA C-terminal glutamine-rich domain and, in addition to other roles and together with previous data, support a role of GAGA as a transcription factor.

Triple-Stranded DNA

The molecular biology of DNA is determined to a great extent by the chemical properties of its constitutive polynucleotide chains and, in particular, by the stability of the different complexes to which they can give rise. Most frequently, DNA is found in the form of an antiparallel doublestranded association but the formation of DNA complexes containing either three or four strands has also been extensively reported in the literature. The formation of a triple-stranded nucleic acid was first reported in 1957 in the case of the RNA triplex U(A · U) (Felsenfeld et al. 1957) and it was followed by the demonstration that the RNA homopolymers polyI and polyA could also form the triple-stranded helixes I(I · I) and I(A · I) (Rich 1958a,b). In the following decade, formation of triple-stranded conformations was also demonstrated for a variety of RNA and DNA homopolymers, as well as for RNA-DNA hybrids. Recently, triple-stranded DNA has received renewed attention. Most of the recent interest in triple-stranded DNA came after the discovery that DNA triplexes could also be intramolecular. In the case of an intramolecular triplex, the third strand, which associates to the double-stranded DNA fragment, is donated by the same DNA molecule. Intramolecular triplexes are therefore a source of DNA structural polymorphism, which adds to the known capability of the DNA molecule to exist under structurally different double-stranded conformations. On the other hand, in the case of an intermolecular triplex, the third strand is donated by a different DNA or RNA molecule, normally a singlestranded oligomer. Intermolecular triplexes provide a means for the specific recognition of double-stranded DNA by single-stranded DNA or RNA molecules. Here, after a brief introductory summary about the general aspects underlying the formation of triple-stranded DNA, we will review the recent progress on the study of the structural and functional properties of intra- and intermolecular DNA triplexes.

Linker histone H1 prevents R-loop accumulation and genome instability in heterochromatin

Linker histone H1 is an important structural component of chromatin that stabilizes the nucleosome and compacts the nucleofilament into higher-order structures. The biology of histone H1 remains, however, poorly understood. Here we show that Drosophila histone H1 (dH1) prevents genome instability as indicated by the increased γH2Av (H2AvS137P) content and the high incidence of DNA breaks and sister-chromatid exchanges observed in dH1-depleted cells. Increased γH2Av occurs preferentially at heterochromatic elements, which are upregulated upon dH1 depletion, and is due to the abnormal accumulation of DNA:RNA hybrids (R-loops). R-loops accumulation is readily detectable in G1-phase, whereas γH2Av increases mainly during DNA replication. These defects induce JNK-mediated apoptosis and are specific of dH1 depletion since they are not observed when heterochromatin silencing is relieved by HP1a depletion. Altogether, our results suggest that histone H1 prevents R-loops-induced DNA damage in heterochromatin and unveil its essential contribution to maintenance of genome stability.While structural importance of linker histone H1 in packaging eukaryotic genome into chromatin is well known, its biological function remains poorly understood. Here the authors reveal that Drosophila linker histone H1 prevents DNA:RNA hybrids accumulation and genome instability in heterochromatin.

General, negative feedback mechanism for regulation of Trithorax-like gene expression in vivo: new roles for GAGA factor in flies

Expression of every gene is first regulated at the transcriptional level. While some genes show acute and discrete periods of expression others show a rather steady expression level throughout development. An example of the latter is Trithorax-like (Trl) a member of the Trithorax group that encodes GAGA factor in Drosophila. Among other functions, GAGA factor has been described to stimulate transcription of several genes, including some homeotic genes. Here we show that GAGA factor is continuously down-regulating the expression of its own promoter using a negative feedback mechanism in vivo. Like its expression, repression by GAGA factor is ubiquitous, prevents its accumulation, and takes place throughout development. Experimental alteration of GAGA factor dosage results in several unexpected phenotypes, not related to alteration of homeotic gene expression, but rather to functions that take place later during development and affect different morphogenetic processes. The results suggest that GAGA factor is essential during development, even after homeotic gene expression is established, and indicate the existence of an upper limit for GAGA factor dosage that should not be exceeded.