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Rolf Ohlsson

Publications and source records attributed to Rolf Ohlsson.

At least 19 recordsLinked to original sources
PubMed2007

A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms' tumourigenesis.

We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis.

CCCTC-Binding Factor↗
PubMed2007

CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

Animals↗
PubMed2006

Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

Animals↗
PubMed2006

CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.

It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.

Animals↗
PubMed2006

The epigenetic progenitor origin of human cancer.

Cancer is widely perceived as a heterogeneous group of disorders with markedly different biological properties, which are caused by a series of clonally selected genetic changes in key tumour-suppressor genes and oncogenes. However, recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of stem/progenitor cells, mediated by 'tumour-progenitor genes'. Furthermore, tumour cell heterogeneity is due in part to epigenetic variation in progenitor cells, and epigenetic plasticity together with genetic lesions drives tumour progression. This crucial early role for epigenetic alterations in cancer is in addition to epigenetic alterations that can substitute for genetic variation later in tumour progression. Therefore, non-neoplastic but epigenetically disrupted stem/progenitor cells might be a crucial target for cancer risk assessment and chemoprevention.

Animals↗
PubMed2005

CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin.

Most of the transcription factors, RNA polymerases and enhancer binding factors are absent from condensed mitotic chromosomes. In contrast, epigenetic marks of active and inactive genes somehow survive mitosis, since the activity status from one cell generation to the next is maintained. For the zinc-finger protein CTCF, a role in interpreting and propagating epigenetic states and in separating expression domains has been documented. To test whether such a domain structure is preserved during mitosis, we examined whether CTCF is bound to mitotic chromatin. Here we show that in contrast to other zinc-finger proteins, CTCF indeed is bound to mitotic chromosomes. Mitotic binding is mediated by a portion of the zinc-finger DNA binding domain and involves sequence specific binding to target sites. Furthermore, the chromatin loop organized by the CTCF-bound, differentially methylated region at the Igf2/H19 locus can be detected in mitosis. In contrast, the enhancer/promoter loop of the same locus is lost in mitosis. This may provide a novel form of epigenetic memory during cell division.

Animals↗
PubMed2005

Familial cases of point mutations in the XIST promoter reveal a correlation between CTCF binding and pre-emptive choices of X chromosome inactivation.

The choice mechanisms that determine the future inactive X chromosome in somatic cells of female mammals involve the regulated expression of the XIST gene. A familial C(-43)G mutation in the XIST promoter results in skewing of X chromosome inactivation (XCI) towards the inactive X chromosome of heterozygous females, whereas a C(-43)A mutation found primarily in the active X chromosome results in the opposite skewing pattern. Both mutations point to the existence of a factor that might be responsible for the skewed patterns. Here we identify this factor as CTCF, a conserved protein with a 11 Zn-finger (ZF) domain that can mediate multiple sequence-specificity and interactions between DNA-bound CTCF molecules. We show that mouse and human Xist/XIST promoters contain one homologous CTCF-binding sequence with the matching dG-contacts, which in the human XIST include the -43 position within the DNase I footprint of CTCF. While the C(-43)A mutation abrogates CTCF binding, the C(-43)G mutation results in a dramatic increase in CTCF-binding efficiency by altering ZF-usage mode required for recognition of the altered dG-contacts of the mutant site. Thus, the skewing effect of the two -43C mutations correlates with their effects on CTCF binding. Finally, CTCF interacts with the XIST/Xist promoter only in female human and mouse cells. The interpretation that this reflected a preferential interaction with the promoter of the active Xist allele was confirmed in mouse fetal placenta. These observations are in keeping with the possibility that the choice of X chromosome inactivation reflects stabilization of a higher order chromatin conformation impinging on the CTCF-XIST promoter complex.

Alleles↗
PubMed2005

Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice.

Loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) is an epigenetic alteration that results in a modest increase in IGF2 expression, and it is present in the normal colonic mucosa of about 30% of patients with colorectal cancer. To investigate its role in intestinal tumorigenesis, we created a mouse model of Igf2 LOI by crossing female H19+/- mice with male Apc+/Min mice. Mice with LOI developed twice as many intestinal tumors as did control littermates. Notably, these mice also showed a shift toward a less differentiated normal intestinal epithelium, reflected by an increase in crypt length and increased staining with progenitor cell markers. A similar shift in differentiation was seen in the normal colonic mucosa of humans with LOI. Thus, altered maturation of nonneoplastic tissue may be one mechanism by which epigenetic changes affect cancer risk.

Adenoma↗
PubMed2005

Poly(ADP-ribosyl)ation and epigenetics. Is CTCF PARt of the plot?

Despite the fact that the poly(ADP-ribose) (PAR) modification of proteins has been known for more than four decades, there is no unifying picture of the pathways governed by this process. While the function of poly(ADP-ribosyl)ation (PARlation) has shown to be traditionally associated with DNA repair and genotoxic stress, there is an emerging view that PARlation is also important in epigenetic regulation of chromatin structure and gene expression in the normal context. This view has been exemplified by the recent demonstration that PARlation is essential for the manifestation of the imprinted state of the Igf2 gene. In particular, the PARlation mark was shown to associate preferentially with the maternally inherited H19 ICR allele, this association depended on functional target sites of the chromatin insulator protein CTCF and, importantly, CTCF itself was found to be PARlated. Given that CTCF is currently the only known factor common for all vertebrate chromatin insulators, it is not surprising that the derepression of the maternal Igf2 allele by 3-aminobenzamide (an inhibitor of PAR polymerases) could be linked to a perturbed chromatin insulator function at the H19 ICR. This feature appears to extend to more than 150 chromatin insulators that were isolated due to their in vivo interaction with CTCF. In this review, we discuss in more depth these results and point out that the turnover of the PARlation mark at DNA-bound CTCF is indicative of a novel mode of rheostat control of expression domains possibly by regulating the stability of higher order chromatin conformations.

Animals↗
PubMed2005

CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator.

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking-Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

Amino Acid Sequence↗
PubMed2004

Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation.

Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.

Animals↗
PubMed2004

The binding sites for the chromatin insulator protein CTCF map to DNA methylation-free domains genome-wide.

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states.

Animals↗
PubMed2004

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region.

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

Base Sequence↗
PubMed2004

Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance.

The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.

Animals↗
PubMed2004

Loss of IGF2 imprinting: mechanisms and consequences.

Although the leading dogma for the origin of the diversity in cancer cell subpopulations is based on a stepwise selection and accumulation of genetic changes that allow uncontrollable malignant growth, there is an emerging understanding that the variability of heritable phenotypes in cancer and cancer-prone cells may also involve epigenetic mechanisms. This is exemplified by the unscheduled activation of the normally repressed maternal IGF2 allele in a wide range of human cancers. The notion that this so-called loss of imprinting (LOI) provides a proliferative advantage by increasing the levels of the IGF-2 ligand available to its receptors is supported by the overgrowth symptoms of neonatal mice, which has been genetically manipulated to express Igf2 biallelically. Although the mechanisms underlying IGF2 LOI in human cancer remains unknown, it is likely to directly or indirectly involve the H19 imprinting control region (ICR). This stretch of 2 kb sequence just upstream of the H19 promoter functions to block enhancer-Igf2 promoter communications by means of a DNA methylation-sensitive chromatin insulator. We discuss here the possibility that pathological mechanisms leading to IGF2 LOI use different strategies with the neutralization of the H19 ICR insulator function as a common denominator.

Animals↗
PubMed2003

The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains.

The repression of the maternally inherited Igf2 allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at the H19 imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenous H19 ICR lead to loss of CTCF binding and Igf2 imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derived H19 ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains.

Animals↗
PubMed2003

Epigenetic variability and the evolution of human cancer.

Although the leading dogma for the origin of the diversity in cancer cell subpopulations is based on a stepwise selection and accumulation of genetic changes that allow uncontrollable malignant growth, there is an emerging understanding that the variability of heritable phenotypes in cancer and cancer-prone cells may also involve epigenetic mechanisms. We discuss here experimental data that allow us to postulate that the genome is organized into epigenetic territories with lineage-specific differences in the stringencies of the active and inactive states. Low-stringency epigenetic states are predicted to be closer to mosaicism, or chaos, than high-stringency states. In pathological situations, the result is an epigenetic variability upon which selection mechanisms can act during tumor progression. This view may have significant implications on clinical assessment and prognosis, and also suggests that major factors involved in the resetting and/or maintenance of epigenetic states may serve as new attractive targets for therapeutic interventions.

Animals↗