R. van Driel

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail and found that the interaction is saturable, of high affinity, and evolutionarily conserved. Competition studies revealed the existence of two different types of interaction related to different structural features of MARs: one involving the minor groove of double-stranded MAR DNA and one involving single-stranded regions. We obtained similar results for the interaction of MARs with intact nuclear matrices from rat liver. A model in which the interaction of nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active state of chromatin is discussed.

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic.

In Drosophila melanogaster, the Polycomb-group (PcG) genes have been identified as repressors of gene expression. They are part of a cellular memory system that is responsible for the stable transmission of gene activity to progeny cells. PcG proteins form a large multimeric, chromatin-associated protein complex, but the identity of its components is largely unknown. Here, we identify two human proteins, HPH1 and HPH2, that are associated with the vertebrate PcG protein BMI1. HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and with BMI1. They also colocalize with BMI1 in interphase nuclei of U-2 OS human osteosarcoma and SW480 human colorectal adenocarcinoma cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic (Ph), and we, therefore, have named the novel proteins HPH1 and HPH2. HPH1, HPH2, and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. Two-hybrid analysis shows that homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts only with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. These data demonstrate the involvement of homology domains I and II in protein-protein interactions and indicate that HPH1 and HPH2 are able to heterodimerize.

The Major Architects of Chromatin: Architectural Proteins in Bacteria, Archaea and Eukaryotes

The genomic DNA of all organisms across the three kingdoms of life needs to be compacted and functionally organized. Key players in these processes are DNA supercoiling, macromolecular crowding and architectural proteins that shape DNA by binding to it. The architectural proteins in bacteria, archaea and eukaryotes generally do not exhibit sequence or structural conservation especially across kingdoms. Instead, we propose that they are functionally conserved. Most of these proteins can be classified according to their architectural mode of action: bending, wrapping or bridging DNA. In order for DNA transactions to occur within a compact chromatin context, genome organization cannot be static. Indeed chromosomes are subject to a whole range of remodeling mechanisms. In this review, we discuss the role of (i) DNA supercoiling, (ii) macromolecular crowding and (iii) architectural proteins in genome organization, as well as (iv) mechanisms used to remodel chromosome structure and to modulate genomic activity. We conclude that the underlying mechanisms that shape and remodel genomes are remarkably similar among bacteria, archaea and eukaryotes.

Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.

Chromocenters in Arabidopsis thaliana are discrete nuclear domains of mainly pericentric heterochromatin. They are characterized by the presence of repetitive sequences, methylated DNA and dimethylated histone H3K9. Here we show that dedifferentiation of specialized mesophyll cells into undifferentiated protoplasts is accompanied by the disruption of chromocenter structures. The dramatic reduction of heterochromatin involves the decondensation of all major repeat regions, also including the centromeric 180 bp tandem repeats. Only the 45S rDNA repeat remained in a partly compact state in most cells. Remarkably, the epigenetic indicators for heterochromatin, DNA methylation and H3K9 dimethylation, did not change upon decondensation. Furthermore, the decondensation of pericentric heterochromatin did not result in transcriptional reactivation of silent genomic elements. The decondensation process was reversible upon prolonged culturing. Strikingly, recondensation of heterochromatin into chromocenters is a stepwise process. Compaction of the tandemly arranged 45S rDNA regions occurs first, followed by the centromeric 180 bp and the 5S rDNA repeats and finally the dispersed repeats, including transposons. The sequence of reassembly seems to be correlated to the size of the repeat domains. Our results indicate that different types of pericentromeric repeats form different types of heterochromatin, which subsequently merge to form a chromocenter.

Interference with the Expression of a Novel Human Polycomb Protein, hPc2, Results in Cellular Transformation and Apoptosis

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.

The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are necessary for 3′‐terminal processing of polyadenylated mRNAs. To study the distribution of 3′ cleavage factors in the nuclei of human T24 cells, monoclonal antibodies against the CstF 64 kDa subunit and against the CPSF 100 kDa subunit were used for immunofluorescent labelling. CstF 64 kDa and CPSF 100 kDa were distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1–4 bright foci. Double immunofluorescence labelling experiments revealed that the foci either overlapped with, or resided next to, a coiled body. Inhibition of transcription with alpha‐amanitin or 5,6‐dichloro‐beta‐D‐ribofuranosyl‐benzimidazole (DRB) resulted in the complete co‐localization of coiled bodies and foci containing 3′ cleavage factors. Electron microscopy on immunogold double‐labelled cells revealed that the foci represent compact spherical fibrous structures, we named ‘cleavage bodies’, intimately associated with coiled bodies. We found that approximately 20% of the cleavage bodies contained a high concentration of newly synthesized RNA, whereas coiled bodies were devoid of nascent RNA. Our results suggest that the cleavage bodies that contain RNA are those that are adjacent to a coiled body. These findings reveal a dynamic and transcription‐dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific transcripts.

Transcriptional Repression Mediated by Polycomb Group Proteins and Other Chromatin-associated Repressors Is Selectively Blocked by Insulators

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophilagypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5′-located DNase I-hypersensitive site in the chicken β-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP

Abstract In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4–90 min at 37°C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br- labeled RNA is a useful technique for high resolution studies of structure–function associations in the nucleolus.

Chromatin folding - from biology to polymer models and back

There is rapidly growing evidence that folding of the chromatin fibre inside the interphase nucleus has an important role in the regulation of gene expression. In particular, the formation of loops mediated by the interaction between specific regulatory elements, for instance enhancers and promoters, is crucial in gene control. Biochemical studies that were based on the chromosome conformation capture (3C) technology have confirmed that eukaryotic genomes are highly looped. Insight into the underlying principles comes from polymer models that explore the properties of the chromatin fibre inside the nucleus. Recent models indicate that chromatin looping can explain various properties of interphase chromatin, including chromatin compaction and compartmentalisation of chromosomes. Entropic effects have a key role in these models. In this Commentary, we give an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics. Starting from simple linear polymer models, we explain how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations. We also discuss different polymer models that describe chromatin folding and compare them to experimental data. Experimentally testing the predictions of such polymer models and their subsequent improvement on the basis of measurements provides a solid framework to begin to understand how our genome is folded and how folding relates to function.