Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

Distinct CoREST complexes act in a cell-type-specific manner.

CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.

Galectin-3 interacts with components of the nuclear ribonucleoprotein complex.

BACKGROUND: The multifunctional ß-galactoside-binding protein galectin-3 is found in many distinct subcellular compartments including the cell nucleus. Expression and distribution of galectin-3 between the cell nucleus and the cytosol changes during cell differentiation and cancer development. Nuclear functions of galectin-3 and how they contribute to tumorigenesis are not understood. METHODS: In order to identify nuclear galectin-3 interaction partners, we used affinity chromatography and co-immunoprecipitation. Spatial proximity in the nucleus was assessed by immunofluorescence and proximity ligation assay. We also investigated the function of galectin-3 on mRNA-export by fluorescence in situ hybridization and on mRNA-processing by RNA-sequencing. RESULTS: The heterogeneous ribonucleoprotein particle component hnRNPA2B1 was identified as a novel galectin-3 binding protein that associates with the lectin in a lactose-dependent manner in the cell nucleus. Specific individual depletion of galectin-3 does not affect the mRNA distribution between cytoplasm and nucleus. A significant alteration of this distribution was observed after combined depletion of galectin-1 and -3. However, silencing of galectin-3 was sufficient to alter the splicing patterns of several genes. CONCLUSIONS: Galectin-3 and hnRNPA2B1 interact as members of the early splicing machinery. Galectin-3 and -1 have redundant functions in mRNA transport and at least in part in mRNA splicing. RNA-sequencing data points to a specific function of the hnRNPA2B1/galectin-3 interaction in the processing of transcripts coding for the nuclear oncoprotein SET.

Identification of SUMO-dependent chromatin-associated transcriptional repression components by a genome-wide RNAi screen.

SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.

LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes.

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.

The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo.

dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.

Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes.

The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.

Stress-induced PARP activation mediates recruitment of Drosophila Mi-2 to promote heat shock gene expression.

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. We show here that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP-dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. We have identified several dMi-2 regions which bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. We suggest that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response.

dMec: a novel Mi-2 chromatin remodelling complex involved in transcriptional repression.

The ATP-dependent chromatin remodeller Mi-2 functions as a transcriptional repressor and contributes to the suppression of cell fates during development in several model organisms. Mi-2 is the ATPase subunit of the conserved Nucleosome Remodeling and Deacetylation (NuRD) complex, and transcriptional repression by Mi-2 is thought to be dependent on its associated histone deacetylase. Here, we have purified a novel dMi-2 complex from Drosophila that is distinct from dNuRD. dMec (dMEP-1 complex) is composed of dMi-2 and dMEP-1. dMec is a nucleosome-stimulated ATPase that is expressed in embryos, larval tissues and adult flies. Surprisingly, dMec is far more abundant than dNuRD and constitutes the major dMi-2-containing complex. Both dNuRD and dMec associate with proneural genes of the achaete-scute complex. However, despite lacking a histone deacetylase subunit, only dMec contributes to the repression of proneural genes. These results reveal an unexpected complexity in the composition and function of Mi-2 complexes.

Conserved mechanisms of NuRD function in hematopoetic gene expression.

The Nucleosome Remodeling and Deacetylating Complex (NuRD) is ubiquitously expressed in all metazoans. It combines nucleosome remodeling and histone deacetylating activities to generate inaccessible chromatin structures and to repress gene transcription. NuRD is involved in the generation and maintenance of a wide variety of lineage-specific gene expression programs during differentiation and in differentiated cells. A close cooperation with a large number of lineage-specific transcription factors is key to allow NuRD to function in many distinct differentiation contexts. The molecular nature of this interplay between transcription factors and NuRD is complex and not well understood. This review uses hematopoiesis as a paradigm to highlight recent advances in our understanding of how transcription factors and NuRD cooperate at the molecular level during differentiation. A comparison of vertebrate and invertebrate systems serves to identify the conserved and fundamental concepts guiding functional interactions between transcription factors and NuRD. We also discuss how the transcription factor-NuRD axis constitutes a potential therapeutic target for the treatment of hemoglobinopathies.

DNA sequence encoded repression of rRNA gene transcription in chromatin.

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes.

RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling.

The ATP-dependent nucleosome remodeler Mi-2/CHD4 broadly modulates chromatin landscapes to repress transcription and to maintain genome integrity. Here we use individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to show that Drosophila Mi-2 associates with thousands of mRNA molecules in vivo. Biochemical data reveal that recombinant dMi-2 preferentially binds to G-rich RNA molecules using two intrinsically disordered regions of unclear function. Pharmacological inhibition of transcription and RNase digestion approaches establish that RNA inhibits the association of dMi-2 with chromatin. We also show that RNA inhibits dMi-2-mediated nucleosome mobilization by competing with the nucleosome substrate. Importantly, this activity is shared by CHD4, the human homolog of dMi-2, strongly suggesting that RNA-mediated regulation of remodeler activity is an evolutionary conserved mechanism. Our data support a model in which RNA serves to protect actively transcribed regions of the genome from dMi-2/CHD4-mediated establishment of repressive chromatin structures.

E2F-Rb complexes regulating transcription of genes important for differentiation and development.

Inactivation of the retinoblastoma tumour suppressor protein (pRb) is a hallmark of most human cancers. Accordingly, pRb is serving as a paradigm in our quest to understand tumour suppressor function. The role played by pRb and the related 'pocket proteins', p107 and p130, in regulating cell cycle progression has been extensively studied over the past two decades. The function of pRb in regulating transcriptional programmes in differentiating cells is less well understood. Recently, the use of a variety of different cell, animal and plant model systems has allowed us a first glimpse at some of the molecular mechanisms underlying pRb-mediated transcriptional regulation during differentiation and development.

Drosophila melanogaster tPlus3a and tPlus3b ensure full male fertility by regulating transcription of Y-chromosomal, seminal fluid, and heat shock genes.

Spermatogenesis in Drosophila melanogaster is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. We addressed how distinct subsets of target genes are selected. We characterized the highly similar proteins tPlus3a and tPlus3b, which contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, we determined the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants using next-generation sequencing (RNA-seq) and compared them to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5, which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. We hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes.

Mass production of Drosophila embryos and chromatographic purification of native protein complexes.

The purification of native protein complexes requires the availability of sufficient amounts of starting material. Drosophila melanogaster embryos have proven to be a rich source for nuclear protein complexes. Here we describe establishment and maintenance of a fly facility for the production of large amounts of embryos, protocols for the production of nuclear extracts, and a scheme for the chromatographic purification of a nuclear multisubunit protein complex.

Tumour-associated missense mutations in the dMi-2 ATPase alters nucleosome remodelling properties in a mutation-specific manner.

ATP-dependent chromatin remodellers are mutated in more than 20% of human cancers. The consequences of these mutations on enzyme function are poorly understood. Here, we characterise the effects of CHD4 mutations identified in endometrial carcinoma on the remodelling properties of dMi-2, the highly conserved Drosophila homologue of CHD4. Mutations from different patients have surprisingly diverse defects on nucleosome binding, ATPase activity and nucleosome remodelling. Unexpectedly, we identify both mutations that decrease and increase the enzyme activity. Our results define the chromodomains and a novel regulatory region as essential for nucleosome remodelling. Genetic experiments in Drosophila demonstrate that expression of cancer-derived dMi-2 mutants misregulates differentiation of epithelial wing structures and produces phenotypes that correlate with their nucleosome remodelling properties. Our results help to define the defects of CHD4 in cancer at the mechanistic level and provide the basis for the development of molecular approaches aimed at restoring their activity.

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-regulated genes.

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins.

The retinoblastoma tumour suppressor in model organisms--new insights from flies and worms.

All forms of life on Earth share a common ancestry. As a consequence, Homo sapiens shares a large number of genes essential for the development and maintenance of multicellular life with "simple" animals, such as the fruit fly Drosophila melanogaster and the nematode worm Caenorhabdites elegans. Indeed, Drosophila and C. elegans have successfully been used to unravel fundamental mechanisms underlying animal development. The sequencing of their genomes has revealed that a surprisingly large proportion of genes relevant for human disease have counterparts in the worm and in the fly. This includes many oncogenes and tumour suppressor genes and provides us with a unique opportunity to exploit the advantages of simple model organisms to further our understanding of the molecular basis of cancer. Recent work on the fly and worm homologs of the Retinoblastoma tumour suppressor (pRb) has uncovered some unexpected pRb functions: Evolutionary conserved pRb complexes participate in cell fate determination, repress germline-specific gene expression and interact with RNA interference pathways. Similar complexes appear to operate in human cells.

Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation.

BACKGROUND: Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. RESULTS: Here we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. CONCLUSIONS: These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.

Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression.

To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.