Rolf Ohlsson

Chromosome crosstalk in three dimensions.

The genome forms extensive and dynamic physical interactions with itself in the form of chromosome loops and bridges, thus exploring the three-dimensional space of the nucleus. It is now possible to examine these interactions at the molecular level, and we have gained glimpses of their functional implications. Chromosomal interactions can contribute to the silencing and activation of genes within the three-dimensional context of the nuclear architecture. Technical advances in detecting these interactions contribute to our understanding of the functional organization of the genome, as well as its adaptive plasticity in response to environmental changes during development and disease.

Does CTCF mediate between nuclear organization and gene expression

The multifunctional zinc‐finger protein CCCTC‐binding factor (CTCF) is a very strong candidate for the role of coordinating the expression level of coding sequences with their three‐dimensional position in the nucleus, apparently responding to a “code” in the DNA itself. Dynamic interactions between chromatin fibers in the context of nuclear architecture have been implicated in various aspects of genome functions. However, the molecular basis of these interactions still remains elusive and is a subject of intense debate. Here we discuss the nature of CTCF‐DNA interactions, the CTCF‐binding specificity to its binding sites and the relationship between CTCF and chromatin, and we examine data linking CTCF with gene regulation in the three‐dimensional nuclear space. We discuss why these features render CTCF a very strong candidate for the role and propose a unifying model, the “CTCF code,” explaining the mechanistic basis of how the information encrypted in DNA may be interpreted by CTCF into diverse nuclear functions.

An Antisense RNA Regulates the Bidirectional Silencing Property of the Kcnq1 Imprinting Control Region

ABSTRACT 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.

Transforming growth factor type β (TGF β) inhibits G1 to S transition, but not activation of human B lymphocytes☆

Abstract Type β transforming growth factor (TGF β) is a polypeptide that may influence the growth of a variety of cell types in a positive or negative fashion. In this study we show that TGF β markedly inhibits DNA synthesis in normal and neoplastic human B lymphocytes stimulated to proliferate with anti-immunoglobulins and B-cell growth factor (BCGF). Although TGF β was needed during the initial 12 h of the culture to promote optimal inhibition, we found that it had little or no effect on several early to intermediate parameters of cell activation ([Ca 2+ ] i increase, c- myc mRNA increase, cellular enlargement, RNA increase, and the increase in the expression of the 4F2 activation antigen). In contrast, TGF β almost completely blocked the induction of transferrin receptor expression, which normally occurs in the late G 1 phase of the cell cycle. Therefore, we conclude that TGF β treatment leads to arrest of the cells in the middle to late G 1 phase, prior to transferrin receptor expression.

CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin

More than 109 base pairs of the genome in higher eucaryotes are positioned in the interphase nucleus such that gene activation, gene repression, remote gene regulation by enhancer elements, and reading as well as adjusting epigenetic marks are possible. One important structural and functional component of chromatin organization is the zinc finger factor CTCF. Two decades of research has advanced the understanding of the fundamental role that CTCF plays in regulating such a vast expanse of DNA.

Replication timing and epigenetic reprogramming of gene expression: a two-way relationship?

An overall link between the potential for gene transcription and the timing of replication in S phase is now well established in metazoans. Here we discuss emerging evidence that highlights the possibility that replication timing is causally linked with epigenetic reprogramming. In particular, we bring together conclusions from a range of studies to propose a model in which reprogramming factors determine the timing of replication and the implementation of reprogramming events requires passage through S phase. These considerations have implications for our understanding of development, evolution and diseases such as cancer.

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 known 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.

Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation

ABSTRACT Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.

Cancer genes, proto-oncogenes, and development

The retroviral cancer genes have in a number of observations been shown to interfere with the developmental program of target cells. Here we are concerned with the interface between cancer genes/proto-oncogenes and developmental processes. Research in this field serves two purposes; to delineate key developmental controls and to identify these as targets for oncogenic agents.

CTCF-binding sites within the H19 ICR differentially regulate local chromatin structures and cis-acting functions

It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5′ and 3′-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3′-region, there is a dramatic gain of long-range chromatin loops impinging on the 5′-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.