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Genomic DNA methylation functions to repress gene expression by interfering with transcription factor binding and/or recruiting repressive chromatin machinery. Recent data support contribution of regulated DNA methylation to embryonic pluripotency, development, and tissue differentiation; this important epigenetic mark is chemically stable yet enzymatically reversible-and heritable through the germline. Importantly, all the major components involved in dynamic DNA methylation are conserved in zebrafish, including the factors that "write, read, and erase" this mark. Therefore, the zebrafish has become an excellent model for studying most biological processes associated with DNA methylation in mammals. Here we briefly review the zebrafish model for studying DNA methylation and describe a series of methods for performing genome-wide DNA methylation analysis. We address and provide methods for methylated DNA immunoprecipitation followed by sequencing (MeDIP-Seq), bisulfite sequencing (BS-Seq), and reduced representation bisulfite sequencing (RRBS-Seq)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cell proliferation and differentiation are guided by changes in gene expression and require the coordinated efforts of the transcription machinery and chromatin-remodeling factors. However, aberrant regulation of chromatin structure can arise through mutations in chromatin-modifying and -remodeling proteins and can lead to improper gene expression and cancer. This review discusses how mutations in chromatin regulators might affect their targeting or activity, with an emphasis on the important insights revealed by leukemogenic fusion proteins. Understanding the normal and oncogenic role of these factors will be crucial for the design of therapeutic agents.

Chromatin remodeling complexes perform central roles in transcriptional regulation. Here, we identify Rsc3 and Rsc30 as novel components of the essential yeast remodeler RSC complex. Rsc3 and Rsc30 function requires their zinc cluster domain, a known site-specific DNA binding motif. RSC3 is essential, and rsc3 Ts- mutants display a G2/M cell cycle arrest involving the spindle assembly checkpoint pathway, whereas rsc30Delta mutants are viable and osmosensitive. Rsc3 and Rsc30 interact functionally and also physically as a stable Rsc3/Rsc30 heteromeric complex. However, DNA microarray analysis with rsc3 or rsc30 mutants reveals different effects on the expression levels of ribosomal protein genes and cell wall genes. We propose that Rsc3 and Rsc30 interact physically but have different roles in targeting or regulating RSC.

RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.

The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

The generation of a local chromatin topology conducive to transcription is a key step in gene regulation. The yeast SWI/SNF complex is the founding member of a family of ATP-dependent remodelling activities capable of altering chromatin structure both in vitro and in vivo. Despite its importance, the pathway by which the SWI/SNF complex disrupts chromatin structure is unknown. Here we use a model system to demonstrate that the yeast SWI/SNF complex can reposition nucleosomes in an ATP-dependent reaction that favours attachment of the histone octamer to an acceptor site on the same molecule of DNA (in cis). We show that SWI/SNF-mediated displacement of the histone octamer is effectively blocked by a barrier introduced into the DNA, suggesting that this redistribution involves sliding or tracking of nucleosomes along DNA, and that it is achieved by a catalytic mechanism. We conclude that SWI/SNF catalyses the redistribution of nucleosomes along DNA in cis, which may represent a general mechanism by which ATP-dependent chromatin remodelling occurs.

The yeast Saccharomyces cerevisiae contains two related chromatin-remodeling complexes, RSC and SWI/SNF, which are shown to share the actin-related proteins Arp7 and Arp9. Depending on the genetic background tested, arp7 delta and arp9 delta mutants are either inviable or show greatly impaired growth and Swi-/Snf- mutant phenotypes. Unlike swi/snf mutants, viable arp7 delta or arp9 delta mutants have an Spt- phenotype, suggesting that RSC affects transcription. Temperature-sensitive mutations in ARP7 and ARP9 were isolated, and the amino acid changes support the structural relationship of Arp7 and Arp9 to actin. However, site-directed mutations predicted to impair ATP binding or hydrolysis did not detectably affect Arp7 or Arp9 function. Our results suggest that actin-related proteins perform important roles in chromatin-remodeling complexes by virtue of structural rather than enzymatic similarities to actin.

RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.

Chromatin is a dynamic material; chromatin structures can repress transcription and their remodeling accompanies activation. Recent biochemical studies in Drosophila have revealed three multi-protein complexes with ATP-dependent chromatin restructuring activities. Although all contain the ATPase ISWI, their properties in vitro are markedly different, distinct from SWI-SNF and reveal intriguing connections to both transcription and chromatin assembly.

A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

We have previously cloned a gene for a developmentally regulated transport protein from the trypanosomatid protozoan Leishmania enriettii. We demonstrate here that this transporter is encoded by a single family of tandemly clustered genes containing approximately 8 copies of the 3.6 kilobase repeat unit. Transcriptional mapping defines a contiguous 3.3 kilobase region of the repeat unit that encodes the mRNA. The 5' end of the mature mRNA contains the spliced leader or mini-exon previously identified in kinetoplastid protozoa, while the 3' ends of the mRNA are heterogeneous in sequence and in location of the polyadenylation site. We have identified genomic restriction fragments that flank the tandem repeat on the 5' and 3' sides and which may be linked to sequences required for expression of the gene family. Other species of Leishmania also contain sequences that hybridize to the cloned L. enriettii gene at high stringency.