Magdalena Murawska

CHD chromatin remodelers and the transcription cycle.

It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by opening or closing chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

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.

dCHD3, a novel ATP-dependent chromatin remodeler associated with sites of active transcription.

ABSTRACT ATP-dependent chromatin remodelers of the CHD family play important roles during differentiation and development. Three CHD proteins, dMi-2, dChd1, and Kismet, have been described for Drosophila melanogaster. Here, we study dCHD3, a novel member of the CHD family. dCHD3 is related in sequence to dMi-2 but lacks several domains implicated in dMi-2 function. We demonstrate that dCHD3 is a nuclear protein and that expression is tightly regulated during fly development. Recombinant dCHD3 remodels mono- and polynucleosomes in an ATP-dependent manner in vitro. Its chromodomains are critical for nucleosome binding and remodeling. Unlike dMi-2, dCHD3 exists as a monomer. Nevertheless, both proteins colocalize with RNA polymerase II to actively transcribed regions on polytene chromosomes, suggesting that both remodelers participate in the process of transcription.

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.

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Background Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. Methodology/Principal Findings We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. Conclusions/Significance Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.

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.

EcR recruits dMi-2 and increases efficiency of dMi-2-mediated remodelling to constrain transcription of hormone-regulated genes

Gene regulation by steroid hormones plays important roles in health and disease. In Drosophila, the hormone ecdysone governs transitions between key developmental stages. Ecdysone-regulated genes are bound by a heterodimer of ecdysone receptor (EcR) and Ultraspiracle. According to the bimodal switch model, steroid hormone receptors recruit corepressors in the absence of hormone and coactivators in its presence. Here we show that the nucleosome remodeller dMi-2 is recruited to ecdysone-regulated genes to limit transcription. Contrary to the prevalent model, recruitment of the dMi-2 corepressor increases upon hormone addition to constrain gene activation through chromatin remodelling. Furthermore, EcR and dMi-2 form a complex that is devoid of Ultraspiracle. Unexpectedly, EcR contacts the dMi-2 ATPase domain and increases the efficiency of dMi-2-mediated nucleosome remodelling. This study identifies a non-canonical EcR-corepressor complex with the potential for a direct regulation of ATP-dependent nucleosome remodelling by a nuclear hormone receptor.

Immunostaining of Drosophila Polytene Chromosomes to Investigate Recruitment of Chromatin-Binding Proteins

Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these factors, including specific interactions with phosphorylated RNA polymerase II and RNA-mediated chromatin associations.

Neoatherosclerosis development following bioresorbable vascular scaffold implantation in diabetic and non-diabetic swine

Background DM remains a risk factor for poor outcome after stent-implantation, but little is known if and how DM affects the vascular response to BVS. Aim The aim of our study was to examine coronary responses to bioresorbable vascular scaffolds (BVS) in swine with and without diabetes mellitus fed a ‘fast-food’ diet (FF-DM and FF-NDM, respectively) by sequential optical coherence tomography (OCT)-imaging and histology. Methods Fifteen male swine were evaluated. Eight received streptozotocin-injection to induce DM. After 9 months (M), 32 single BVS were implanted in epicardial arteries with a stent to artery (S/A)-ratio of 1.1:1 under quantitative coronary angiography (QCA) and OCT guidance. Lumen, scaffold, neointimal coverage and composition were assessed by QCA, OCT and near-infrared spectroscopy (NIRS) pre- and/or post-procedure, at 3M and 6M. Additionally, polarization-sensitive (PS)-OCT was performed in 7 swine at 6M. After sacrifice at 3M and 6M, histology and polymer degradation analysis were performed. Results Late lumen loss was high (~60%) within the first 3M after BVS-implantation (P<0.01 FF-DM vs. FF-NDM) and stabilized between 3M and 6M (<5% change in FF-DM, ~10% in FF-NDM; P>0.20). Neointimal coverage was highly heterogeneous in all swine (DM vs. NDM P>0.05), with focal lipid accumulation, irregular collagen distribution and neointimal calcification. Likewise, polymer mass loss was low (~2% at 3M, ~5% at 6M;P>0.20) and not associated with DM or inflammation. Conclusion Scaffold coverage showed signs of neo-atherosclerosis in all FF-DM and FF-NDM swine, scaffold polymer was preserved and the vascular response to BVS was not influenced by diabetes.