Chromatin Restructuring Research Articles

The X and Y Chromosomes Assemble into H2A.Z, Containing Facultative Heterochromatin, following Meiosis

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.

MYST family histone acetyltransferases in the protozoan parasite Toxoplasma gondii.

The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a approximately 3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is approximately 7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.

In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae

The autonomously replicating sequence-binding factor 1 (ABF1) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABF1 binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5′-CGTnnnnnnnGA(G/C)-3′. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABF1. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABF1. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABF1 with nearly two-magnitude higher affinity as compared to the hitherto accepted ABF1 consensus sequence. This led us to postulate a strong recognition motive: 5′-TnnCGTnnnnnnTGAT-3′.

Causes and consequences of DNA hypomethylation in human cancer

While specific genes are hypermethylated in the genome of cancer cells, overall methylcytosine content is often decreased as a consequence of hypomethylation affecting many repetitive sequences. Hypomethylation is also observed at a number of single-copy genes. While global hypomethylation is highly prevalent across all cancer types, it often displays considerable specificity with regard to tumor type, tumor stage, and sequences affected. Following an overview of hypomethylation alterations in various cancers, this review focuses on 3 hypotheses. First, hypomethylation at a single-copy gene may occur as a 2-step process, in which selection for gene function follows upon random hypo methylation. In this fashion, hypomethylation facilitates the adaptation of cancer cells to the ever-changing tumor tissue microenvironment, particularly during metastasis. Second, the development of global hypomethylation is intimately linked to chromatin restructuring and nuclear disorganization in cancer cells, reflected in a large number of changes in histone-modifying enzymes and other chromatin regulators. Third, DNA hypomethylation may occur at least partly as a consequence of cell cycle deregulation disturbing the coordination between DNA replication and activity of DNA methyltransferases. Finally, because of their relation to tumor progression and metastasis, DNA hypomethylation markers may be particularly useful to classify cancer and predict their clinical course.

Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor

We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

Remodeling Chromatin in the Biology and Treatment of Acute Leukemia

Therapy for acute leukemia has changed markedly over the past decade with the introduction of oncogene targeted therapy for certain subtypes, including acute promyelocytic leukemia (APML) and Philadelphia chromosome positive acute lymphoblastic leukemia, and better utilization of the graft-versus-leukemia effect in allotransplantation. New discoveries regarding the regulation of transcription by chromatin remodeling complexes has furthered our understanding of leukemia biology and shed light on the mechanisms behind some of our current therapies such as all-trans retinoic acid. Unlike chronic myeloid leukemia in which deregulation of a specific tyrosine kinase is responsible for driving the disease process, the majority of oncogenes involved in the pathogenesis of acute leukemia are abnormal fusion proteins acting as de-regulated transcription factors that prevent normal gene expression programs via chromatin mediated silencing. Many of these transcription factors are highly regulated in certain developmental compartments in normal physiology, but when aberrantly expressed, can block the differentiation of progenitor cells leading to leukemia. Targeted therapy with all trans-retinoic acid in acute promyelocytic leukemia, for example, disrupts the recruitment of histone deacetylases to transcriptional complexes and allows normal myeloid maturation to proceed. Ongoing studies into the biology of leukemia suggest that despite the number and diversity of cytogenetic abnormalities detected, leukemogenesis depends on a relatively small number of abnormal signaling pathways (such as the de-repression of core binding factor activity), many of which are involved in the restructuring of chromatin. Targeted therapy directed against specific chromatin remodifying activity offers great potential in the treatment of other subtypes of acute leukemia. Less specific chromatin remodeling therapies such as the histone deacetylase inhibitors which include valproate, trichomans A, thalidomide and butyrate have anti-leukemic activity in vitro in non-ATRA responsive leukemias and clinical trials with such agents are currently proceeding.

Herpes simplex virus 1 U(L)31 and U(L)34 gene products promote the late maturation of viral replication compartments to the nuclear periphery.

Herpes simplex virus 1 (HSV-1) forms replication compartments (RCs), domains in which viral DNA replication, late-gene transcription, and encapsidation take place, in the host cell nucleus. The formation of these domains leads to compression and marginalization of host cell chromatin, which forms a dense layer surrounding the viral RCs and constitutes a potential barrier to viral nuclear egress or primary envelopment at the inner nuclear membrane. Surrounding the chromatin layer is the nuclear lamina, a further host cell barrier to egress. In this study, we describe an additional phase in RC maturation that involves disruption of the host chromatin and nuclear lamina so that the RC can approach the nuclear envelope. During this phase, the structure of the chromatin layer is altered so that it no longer forms a continuous layer around the RCs but instead is fragmented, forming islands between which RCs extend to reach the nuclear periphery. Coincident with these changes, the nuclear lamina components lamin A/C and lamin-associated protein 2 appear to be redistributed via a mechanism involving the U(L)31 and U(L)34 gene products. Viruses in which the U(L)31 or U(L)34 gene has been deleted are unable to undergo this phase of chromatin reorganization and lamina alterations and instead form RCs which are bounded by an intact host cell chromatin layer and nuclear lamina. We postulate that these defects in chromatin restructuring and lamina reorganization explain the previously documented growth defects of these mutant viruses.

Replicating by the clock.

The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.

Arabidopsis contains ancient classes of differentially expressed actin-related protein genes.

Actin-related proteins (ARPs) share less than 60% amino acid sequence homology with conventional actins and have roles in diverse cytoskeletal processes in the cytoplasm and nucleus. The genome of Arabidopsis was explored for possible ARP gene family members. Eight potential ARP gene sequences were found dispersed on three of the five Arabidopsis chromosomes. AtARP2 and AtARP3 are protein orthologs of their similarly named counterparts in other kingdoms. AtARP4, AtARP5, and AtARP6 are orthologs of two classes of nuclear ARPs previously characterized in animals and fungi, BAF53s and ARP6s. AtARP7 and AtARP8 appear to be novel proteins that are not closely related to any known animal or fungal ARPs, and may be plant specific. The complex Arabidopsis ARP gene structures each contain from five to 20 exons. Expressed transcripts were identified and characterized for AtARP2 through AtARP8, but not for AtARP9, and transcripts representing two splice variants were found for AtARP8. The seven expressed genes are predicted to encode proteins ranging from 146 to 471 amino acids in length. Relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are expressed at very low levels in all organs. AtARP5, AtARP6, and AtARP8 each have distinct transcript expression patterns in seedlings, roots, leaves, flowers, and siliques. Using isovariant-specific monoclonal antibodies, AtARP4 and AtARP7 proteins were shown to be most highly expressed in flowers. The likely involvement of plant ARPs in actin nucleation, branching of actin filaments, chromatin restructuring, and transcription are briefly discussed.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

Chromatin alteration, transcription and replication: What's the opening line to the story?

Polymerase accessibility to chromatin is a limiting step in both RNA and DNA synthesis. Unwinding DNA and nucleosomes during polymerase complex binding and processing likely requires priming by chromatin restructuring. The initiating step in these processes remains an area of speculation. This review focuses on the physical handling of chromatin during transcription and replication, the fate of nucleosomes assembled on DNA during unwinding and processing the chromatin substrate, and how these alterations in chromatin structure may affect gene expression. Transcription or replication may alter chromatin structure during synthesis, enabling regulatory factor binding and, potentially, future rounds of transcription. As chromatin remodeling and transcription factor binding augment transcription and replication, and are themselves increased by these processes, a temporal model of structural alterations and gene activation is built that may be more circular than linear.

The Drosophila Brahma complex is an essential coactivator for the trithorax group protein Zeste

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

Ikaros DNA-Binding Proteins Direct Formation of Chromatin Remodeling Complexes in Lymphocytes

The Ikaros gene family encodes zinc finger DNA-binding proteins essential for lineage determination and control of proliferation in the lymphoid system. Here, we report that, in the nucleus of a T cell, a major fraction of Ikaros and Aiolos proteins associate with the DNA-dependent ATPase Mi-2 and histone deacetylases, in a 2 MD complex. This Ikaros–NURD complex is active in chromatin remodeling and histone deacetylation. Upon T cell activation, Ikaros recruits Mi-2/HDAC to regions of heterochromatin. These studies reveal that Ikaros proteins are capable of targeting chromatin remodeling and deacetylation complexes in vivo. We propose that the restructuring of chromatin is a key aspect of Ikaros function in lymphocyte differentiation.

Persistent site-specific remodeling of a nucleosome array by transient action of the SWI/SNF complex.

The SWI/SNF complex participates in the restructuring of chromatin for transcription. The function of the yeast SWI/SNF complex in the remodeling of a nucleosome array has now been analyzed in vitro. Binding of the purified SWI/SNF complex to a nucleosome array disrupted multiple nucleosomes in an adenosine triphosphate-dependent reaction. However, removal of SWI/SNF left a deoxyribonuclease I-hypersensitive site specifically at a nucleosome that was bound by derivatives of the transcription factor Gal4p. Analysis of individual nucleosomes revealed that the SWI/SNF complex catalyzed eviction of histones from the Gal4-bound nucleosomes. Thus, the transient action of the SWI/SNF complex facilitated irreversible disruption of transcription factor-bound nucleosomes.

The Testis-Specific High-Mobility-Group Protein, a Phosphorylation-Dependent DNA-Packaging Factor of Elongating and Condensing Spermatids

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.

Localization of acetylated histone H4 in the macronucleus of Euplotes

Antisera specific to acetylated and unacetylated N-terminal domains of histone H4 were employed to map the spatial distribution of exposed antigenic determinants in the macronucleus of Euplotes eurystomus. Sites of putative transcription-related acetylation appear to reside in the regions between condensed chromatin granules and disappear during chromatin restructuring in the forward zone of replication bands. Coincident with synthesis of newly replicated chromatin, the specific antisera reveal a resumption of exposed sites of H4 acetylation in the rear zone of replication bands.

Time dependence of X gene reactivation induced by 5-azacytidine: Possible progressive restructuring of chromatin

According to the theoretical mechanism of DNA demethylation by 5-azacytidine, the complete demethylation of one site will require two cell divisions. If reexpression is directly related to demethylation, a maximal reexpression is expected after two cell divisions. In a hamster X human hybrid cell line containing an inactive human X chromosome treated by 5-azacytidine, we show that HPRT reactivation frequency is increased more than 10-fold when cells are allowed to divide 14 times before the selection for the HPRT reactivants. We suggest that the delay corresponds to changes in chromatin conformation.