Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

p53 regulates the mevalonate pathway in human glioblastoma multiforme.

The mevalonate (MVA) pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, we provided evidence that p53 induces the expression of a group of enzymes of the MVA pathway including 3'-hydroxy-3'-methylglutaryl-coenzyme A reductase, MVA kinase, farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, in the human glioblastoma multiforme cell line, U343 cells, and in normal human astrocytes, NHAs. Genetic and pharmacologic perturbation of p53 directly influences the expression of these genes. Furthermore, p53 is recruited to the gene promoters in designated p53-responsive elements, thereby increasing their transcription. Such effect was abolished by site-directed mutagenesis in the p53-responsive element of promoter of the genes. These findings highlight another aspect of p53 functions unrelated to tumor suppression and suggest p53 as a novel regulator of the MVA pathway providing insight into the role of this pathway in cancer progression.

ZRF1 controls the retinoic acid pathway and regulates leukemogenic potential in acute myeloid leukemia.

Acute myeloid leukemia (AML) is frequently linked to epigenetic abnormalities and deregulation of gene transcription, which lead to aberrant cell proliferation and accumulation of undifferentiated precursors. ZRF1, a recently characterized epigenetic factor involved in transcriptional regulation, is highly overexpressed in human AML, but it is not known whether it plays a role in leukemia progression. Here, we demonstrate that ZRF1 depletion decreases cell proliferation, induces apoptosis and enhances cell differentiation in human AML cells. Treatment with retinoic acid (RA), a differentiating agent currently used to treat certain AMLs, leads to a functional switch of ZRF1 from a negative regulator to an activator of differentiation. At the molecular level, ZRF1 controls the RA-regulated gene network through its interaction with the RA receptor α (RARα) and its binding to RA target genes. Our genome-wide expression study reveals that ZRF1 regulates the transcription of nearly half of RA target genes. Consistent with our in vitro observations that ZRF1 regulates proliferation, apoptosis, and differentiation, ZRF1 depletion strongly inhibits leukemia progression in a xenograft mouse model. Finally, ZRF1 knockdown cooperates with RA treatment in leukemia suppression in vivo. Taken together, our data reveal that ZRF1 is a key transcriptional regulator in leukemia progression and suggest that ZRF1 inhibition could be a novel strategy to be explored for AML treatment.

ZRF1 controls oncogene-induced senescence through the INK4-ARF locus.

The reactivation of the INK4-ARF locus, which is epigenetically repressed by Polycomb proteins in healthy cells, is a hallmark of senescence. One mechanism of reactivating Polycomb-silenced genes is mediated by the epigenetic factor ZRF1, which associates with ubiquitinated histone H2A. We show that cells undergoing senescence following oncogenic Ras expression have increased ZRF1 levels, and that this binds to the p15INK4b, ARF and p16INK4a promoters. Furthermore, ZRF1 depletion in oncogenic Ras-expressing cells restores proliferation by preventing Arf and p16Ink4a expression, consequently bypassing senescence. Thus, ZRF1 regulates the INK4-ARF locus during cellular proliferation and senescence, and alterations in ZRF1 may contribute to tumorigenesis.

Roles of the Polycomb group proteins in stem cells and cancer.

Polycomb group proteins have long been linked to the occurrence of different forms of cancer. Polycomb proteins form at least two distinct complexes, the Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2). Some of the PRC complex subunits have been found to be overexpressed in a variety of different tumors. Epigenetic perturbations are likely to be the cause for transcriptional misregulation of tumor suppressor genes and of certain cell fates. It is especially critical for stem cells that their potential to self-renewal and to differentiate is tightly controlled and properly orchestrated. Misregulation of Polycomb protein levels often leads to either a block or unscheduled activation of developmental pathways, thereby enhancing the proliferation capability of a cell. The consequences of this misregulation have been linked to the establishment of cancer stem cells, which can produce tumors through a combination of increased self-renewal and the lack of complete cellular differentiation. Cancer stem cells are believed to persist within tumors and to elicit relapse and metastasis. In this review, we recapitulate the roles of Polycomb proteins in stem cell biology, and the impact their misregulation can have on cancer.

PML4 induces differentiation by Myc destabilization.

Opposing functions like oncogene and tumor suppressions have been established for c-Myc and promyelocytic leukemia (PML) protein, respectively. Myc is known to inhibit differentiation of hematopoietic precursor cells, and here we report that PML promotes cell differentiation. We further demonstrate that PML and Myc form a complex in vivo. The interaction of the two proteins leads to the destabilization of Myc in a manner dependent on the really interesting new gene (RING) domain of PML. Although several PML isoforms are able to interact with Myc, the ability to destabilize Myc is specific for PML4. Importantly, the PML-induced destabilization resulted in a reduction of promoter-bound Myc on Myc-repressed genes. Thereby, PML induced the re-activation of Myc-repressed target genes including the tumor suppressive genes of the cell cycle inhibitors cdkn1a/p21 and cdkn2b/p15. Together, these results establish PML-mediated destabilization of Myc and the derepression of cell cycle inhibitor genes as an important regulatory mechanism that allows cell differentiation and prevents aberrant proliferation driven by uncontrolled Myc activity.

Chromatin structure and epigenetics.

In eukaryotic cells, the DNA molecule is found in the form of a nucleoprotein complex named chromatin. The basic unit of the chromatin is the nucleosome, which comprises 147 base pairs of DNA wrapped around an octamer of core histones (made of two molecules of each H2A, H2B, H3, and H4 histones). Each nucleosome is linked to the next by small segments of linker DNA. Most chromatin is further condensated by winding in a polynucleosome fibre, which may be stabilized through the binding of histone H1 to each nucleosome and to the linker DNA. The modulation of the structure of the chromatin fibre is critical for the regulation of gene expression since it determines the accessibility and the sequential recruitment of regulatory factors to the underlying DNA. Depending on the different transcriptional states, the structure of the chromatin may be altered in its constituents (e.g. the presence of repressors, activators, chromatin remodelling complexes, and/or incorporation of histone variants), and in covalent modifications of its constituents (such as DNA methylation at cytosine residues, and posttranslational modifications of histone tails). Here, we give an overview of the molecular mechanisms involved in chromatin regulation and the epigenetic transmission of its state, both in normal and pathological scenarios.

Epigenetic gene silencing in acute promyelocytic leukemia.

The recent explosion in our knowledge of how chromatin organization modulates gene transcription has highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes--in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing--affect virtually every step in tumor progression. Intriguingly, methylation patterns are severely altered in tumors, with an overall hypomethylation of the genome and hypermethylation of islands of CpGs clusters within specific DNA regions. Though overexpression of DNA methyltransferases (DNMTs) has been proposed to be a mechanism for aberrant genome methylation, it does not explain the specific regional hypermethylation in cancer cells. We have analyzed the role of chromatin modifying activities in cell transformation using acute promyelocytic leukemia as a model system. This disease is caused by expression of the PML-RARalpha fusion protein, thus offering the opportunity of studying the mechanisms of leukemogenesis through molecular investigation of the activity of the directly transforming protein. Recent evidence suggests that PML-RARalpha as well as other leukemia-associated fusion proteins induce changes in the chromatin structure. Specifically, aberrant recruitment of different chromatin modifying enzymes to specific promoters induces DNA hypermethylation and heterochromatin formation, which consequentially leads to the transcriptional silencing of that genes. Importantly, these epigenetic modifications were found to contribute to the leukemogenic potential of PML-RARalpha. These observations suggest that epigenetic alterations could actively contribute to the development of APL and other hyperproliferative diseases.

Effects of the acute myeloid leukemia--associated fusion proteins on nuclear architecture.

Acute myeloid leukemias (AMLs) are consistently associated with chromosomal rearrangements that result in the generation of chimeric genes and fusion proteins. One of the two affected genes is frequently a transcription factor Involved in the regulation of hematopoletic differentiation. Recent findings suggest a common leukemogenic mechanism for the fused transcription factor: abnormal recruitment of histone deacetylase (HDAC)-containing complexes to its target promoters. Inhibition of HDAC enzymatic activity reverts the leukemic phenotype in vitro and therefore represents a plausible strategy for antileukemic therapy. In this review, we first briefly describe the molecular structure and mechanisms of the most frequent AML associated fusion proteins (RAR, MLL, and CBF fusions) and then summarize available knowledge about their effects on the nuclear architecture. We propose that alteration of nuclear compartmentalization might represent an additional common mechanism of leukemogenesis.

Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Two-step synergism between the progesterone receptor and the DNA-binding domain of nuclear factor 1 on MMTV minichromosomes.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

Rapid purification of intact minichromosomes over a glycerol cushion.

Negatively supercoiled plasmids can be assembled into dynamic minichromosomes using Drosophila embryo extract as a source of histones and chromatin assembly factors. However, analysis of such mini-chromosomes is often difficult due to the presence in the crude extract of a large excess of macromolecules and low molecular weight molecules including ATP. Several techniques have been used to partially purify the minichromosomes based on either sizing columns or centrifugation on sucrose gradients. We have developed a single-step method employing a 30 min ultracentrifugation through a glycerol cushion. In contrast to chromatin purified in sucrose-containing buffers, the minichromosomes obtained with this method are suitable for transcriptional analysis. This method is fast, quantitative, flexible, can deal with several samples simultaneously and leads to concentration of the chromatin. As centrifugation through glycerol yields chromatin free of ATP and several characterized chromatin remodeling complexes, this method should be useful for structural and functional studies in vitro.

The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region.

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.

Estrogen stimulates intracellular traffic in the liver of Rana esculenta complex by modifying Rab protein content.

During vitellogenesis in oviparous animals, estrogens induce the synthesis of the yolk precursor vitellogenin, a lipophosphoprotein rich in cholesterol. Estrogens also induce the activity of 3-hydroxy-3-methylglutaryl CoA reductase, that is necessary for the lipidation of vitellogenin. This increased enzyme activity could also be important for the production of isoprenoid groups that post-translationally modify proteins such as the Rab proteins, which are small G proteins involved in intracellular traffic. The effect of estrogens on the production of prenylated proteins and on the levels of Rab proteins in the liver of Rana esculenta complex has been studied. An increase of the Rabs specifically involved in the exocytic pathway was observed and is probably related to the need for export of massive amounts of newly synthesized vitellogenin.

Assembly of MMTV promoter minichromosomes with positioned nucleosomes precludes NF1 access but not restriction enzyme cleavage.

To generate long arrays of nucleosomes within a topologically defined chromatin domain we have assembled minichromosomes on negatively supercoiled plasmid DNA with extracts from Drosophila preblastoderm embryos. These minichromosomes are dynamic substrates for energy-dependent nucleosome remodeling machines that facilitate the binding of various transcription factors but do not exhibit nucleosome positioning. In contrast, if such minichromosomes include the mouse mammary tumour virus (MMTV) promoter we find it wrapped around a nucleosome with similar translational and rotational position as in vivo . This structure precluded binding of NF1 to its cognate site at -75/-65 at salt concentrations between 60 and 120 mM, even in the presence of ATP, which rendered the NF1 site accessible to the restriction enzyme Hin fI. However, insertion of 30 bp just upstream of the NF1 site, which moves the site to the linker DNA, allowed ATP-dependent binding of NF1 to a fraction of the minichromosomes, even in the presence ofstoichiometric amounts of histone H1. The minichromosomes assembled in the Drosophila embryo extract reproduce important features of the native MMTV promoter chromatin and reveal differences in the ability of transcription factors and restriction enzymes to access their binding sites in positioned nucleosomes.

Nuclear lamina assembly in the first cell cycle of rat liver regeneration.

The nuclear lamina is a fibrous structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of its protein components, the lamins A, B, and C. The isoprenylation of the lamins is essential for their proper membrane anchoring and functionality. The content and the membrane association of nuclear lamins and the subcellular localization at light and electron microscopical levels were studied at different times of rat liver regeneration. This model for the good synchrony of the first cell cycle is particularly suited for the study of cell-cycle-dependent modifications and is particularly interesting for the increased protein prenylation found in S phase. The biochemical results show an increased lamin content in nuclear proteins in G1 phase and a decreased content in M phase, along with an enhanced cytosolic localization of A and C lamins at later stages. The morphological results show in M phase, also in nondividing cells, a decreased lamin-like immunoreactivity around the nucleus with an apparent nuclear lamina disassembly. These data emphasize the dynamic organization of nuclear lamina not only in mitosis but also in interphase. The reduction and partial solubilization of nuclear lamina in M phase suggest a reorganization of the nuclear envelope also in those cells that do not appear in mitosis but have replicated their DNA content that will result in a higher degree of polyploidy.

Independent responsiveness of frog liver low-density lipoprotein receptor and HMGCoA reductase to estrogen treatment.

Cholesterol metabolism in the female frog exhibits circannual modifications which parallel plasma estrogen fluctuations. Estrogens enhance production and lipidation of the yolk precursor vitellogenin by inducing the transcription of its gene and by stimulating the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. The time dependence of the effects that these hormones have on HMGCoA reductase and the low-density lipoprotein (LDL) receptor in the liver of Rana esculenta complex has been investigated. Following estrogen treatment, the levels of LDL receptor mRNA and protein gradually increased, with a maximum concentration observed at 3 days. The HMGCoA reductase protein level increased progressively, while the mRNA level was not significantly modified. Thus the LDL receptor and HMGCoA reductase in frog behave independently after estrogen stimulation, as already reported to occur in the rat. This suggests a uncoordinated regulation that might be even partially related with the changes in cellular cholesterol content.