Yurii Sedkov

Transcription of bxd Noncoding RNAs Promoted by Trithorax Represses Ubx in cis by Transcriptional Interference

Much of the genome is transcribed into long noncoding RNAs (ncRNAs). Previous data suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex (BX-C) prevent silencing of Ultrabithorax (Ubx) and recruit activating proteins of the trithorax group (trxG) to their maintenance elements (MEs). We found that, surprisingly, Ubx and several bxd ncRNAs are expressed in nonoverlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. Our data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. We propose that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription.

TrxG and PcG Proteins but Not Methylated Histones Remain Associated with DNA through Replication

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.

Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site (reviewed in ref. 3). The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. Here, we report a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5′-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5′-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation.

Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. Here we report that TRR is a histone methyltransferases capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. We propose that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters.

Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

ABSTRACT In Drosophila, two classes of genes, thetrithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within thebxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each ∼400 bp) contain both TRX- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenousUltrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (∼90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group genepleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators.

Transcriptional interference: an unexpected layer of complexity in gene regulation

Much of the genome is transcribed into long untranslated RNAs, mostly of unknown function. Growing evidence suggests that transcription of sense and antisense untranslated RNAs in eukaryotes can repress a neighboring gene by a phenomenon termed transcriptional interference. Transcriptional interference by the untranslated RNA may prevent recruitment of the initiation complex or prevent transcriptional elongation. Recent work in yeast, mammals, and Drosophila highlights the diverse roles that untranslated RNAs play in development. Previously, untranslated RNAs of the bithorax complex of Drosophila were proposed to be required for its activation. Recent studies show that these untranslated RNAs in fact silence Ultrabithorax in early embryos, probably by transcriptional interference.

Ecdysone- and NO-Mediated Gene Regulation by Competing EcR/Usp and E75A Nuclear Receptors during Drosophila Development

The Drosophila ecdysone receptor (EcR/Usp) is thought to activate or repress gene transcription depending on the presence or absence, respectively, of the hormone ecdysone. Unexpectedly, we found an alternative mechanism at work in salivary glands during the ecdysone-dependent transition from larvae to pupae. In the absense of ecdysone, both ecdysone receptor subunits localize to the cytoplasm, and the heme-binding nuclear receptor E75A replaces EcR/Usp at common target sequences in several genes. During the larval-pupal transition, a switch from gene activation by EcR/Usp to gene repression by E75A is triggered by a decrease in ecdysone concentration and by direct repression of the EcR gene by E75A. Additional control is provided by developmentally timed modulation of E75A activity by NO, which inhibits recruitment of the corepressor SMRTER. These results suggest a mechanism for sequential modulation of gene expression during development by competing nuclear receptors and their effector molecules, ecdysone and NO.

Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein.

The products of the trithorax and Polycomb groups genes maintain the activity and silence, respectively, of many developmental genes including genes of the homeotic complexes. This transcriptional regulation is likely to involve modification of chromatin structure. Here, we report the cloning and characterization of a new gene, trithorax-related (trr), which shares sequence similarities with members of both the trithorax and Polycomb groups. The trr transcript is 9.6 kb in length and is present throughout development. The TRR protein, as predicted from the nucleotide sequence of the open reading frame, is 2431 amino acids in length and contains a PHD finger-like domain and a SET domain, two highly conserved protein motifs found in several trithorax and Polycomb group proteins, and in modifiers of position effect variegation. TRR is most similar in sequence to the human ALR protein, suggesting that trr is a Drosophila homologue of the ALR. TRR is also highly homologous to Drosophila TRITHORAX protein and to its human homologue, ALL-1/HRX. However, preliminary genetic analysis of a trr null allele suggests that TRR protein may not be involved in regulation of homeotic genes (i.e. not a member of the trithorax or Polycomb groups) or in position effect variegation.

Association of trxG and PcG proteins with the bxd maintenance element depends on transcriptional activity

Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes can antagonize each others function, whereas mutations of genes within each group have synergistic effects. Here, we show in Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and Asx require Trithorax in order to bind to their target genes. Together, our data argue that at the single-cell level, association of repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically.

A Model for Initiation of Mosaic HOX Gene Expression Patterns by Non-Coding RNAs in Early Embryos

There is growing appreciation for the role of non-coding (nc) RNA in regulation of HOX genes of Drosophila. Our data suggest that current models for activation by ncRNA at the bithorax complex (BX-C) genes are mistaken. We propose that bxd and iab ncRNAs repress coding HOX genes Ultrabithorax and abdominal-A, respectively, by transcriptional interference. It is not clear how regulation by non-coding RNAs is integrated with other regulatory mechanisms at HOX loci. We suggest that non-coding RNAs regulated by the trithorax group of epigenetic regulators have an early transient role in repression of HOX genes at the bithorax complex. Later, we propose that repression by HOX proteins, and members of the Polycomb group take over from repression by ncRNAs. We discuss emerging research questions in light of this model.