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Briefings in Functional Genomics and Proteomics Advance Access originally published online on February 23, 2006
Briefings in Functional Genomics and Proteomics 2006 5(1):24-31; doi:10.1093/bfgp/ell003
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Special Issues Papers

The positive aspects of stress: strain initiates domain decondensation (SIDD)

Silke Winkelmann, Martin Klar, Craig Benham, AK Prashanth, Sandra Goetze, Angela Gluch and Juergen Bode

Corresponding author. Juergen Bode, GBF German Research Center for Biotechnology/Epigenetic Regulation Mascheroder Weg 1, D-38124 Braunschweig, Germany. Tel: +49 (531) 6181 251; Fax: +49 (531) 6181 262. E-mail: jbo{at}gbf.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PERSPECTIVE
 References
 
The conventional string-based bioinformatic methods of genomic sequence analysis are often insufficient to identify DNA regulatory elements, since many of these do not have a recognizable motif. Even in case a sequence pattern is known to be associated with an element it may only partially mediate its function. This suggests that properties not correlated with the details of base sequence contribute to regulation. One of these attributes is the DNA strand-separation potential, known as SIDD (stress-induced duplex destabilization) which facilitates the access of tracking proteins and the formation of local secondary structures. Using the type 1 interferon gene cluster as a paradigm, we demonstrate that the imprints in a SIDD profile coincide with chromatin domain borders and with DNAse I hypersensitive sites to which regulatory potential could be assigned. The approach permits the computer-guided identification of yet unknown, mostly remote sites and the design of artificial elements with predictable properties for multiple applications.

Keywords: chromatin domains, interferon gene cluster, remote control elements, non-viral episomes, SIDD, DNAse I hypersensitive sites


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PERSPECTIVE
 References
 
The effects of DNA superhelicity have first been studied in circular plasmids, which naturally occur in an underwound (i.e. negatively supercoiled) rather than in a relaxed state. Using pBR322 DNA as a model, Mung bean nuclease digestion experiments have shown strand separation at two regulatory sites, the promoter region and the 3' terminus of the ampicillin resistance gene [1]. Starting with this model the SIDD-algorithm has been developed and continuously extended to provide a universal tool to predict the location of ‘base-unpairing regions’ (BURs) where strand separation occurs under superhelical strain and in a wider sequence context (Figure 1B'). The algorithm is based on a statistical mechanical procedure that has been outlined previously [2, 3].


Figure 1
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  		Figure 1: Structural features along the human type I IFN gene cluster. (A) The cluster on the short arm of human chromosome 9. Solid arrowheads mark functional genes while open arrowheads indicate pseudogenes. Vertical arrows stand for the position of UEs/DNAse hypersensitive sites. Black boxes below the line symbolize strong S/MARs while open boxes stand for tested regions without major S/MAR character. A172 and A1235 are fragile sites (deletion endpoints) adjacent to S/MAR elements. (B') SIDD-profile for the IFNB1 gene domain. A SIDD profile shows the propensity of a base pair to separate under negative superhelical tension ({sigma} = –0.05) and in a given sequence context. Sites preferred for strand separation have a G < 0 kcal/mol. The slightly destabilized B1 promoter and the pronounced B1 terminator are indicated upstream and downstream from the coding-sequence (horizontal arrow). The strongly destabilized main peaks HS1, HS2 and HS3 represent upstream control elements [9, 10]. (B'') SIDD structure of the IFNB1-upstream S/MAR, analysed as part of a pUVC-type vector. Since all UEs are in competition with one another, the over-all destabilization of the S/MAR can be estimated from the size of the AmpT and AmpP sites in the vector backbone. (C) Functional genes (IFNA10 and IFNA7) and a pseudogene (IFNWP18) within subcluster III. Note that the pseudogene has totally lost a destabilized promoter region and that destabilization at the downstream end is largely reduced. Between IFNA10 and IFNA7 a potential regulatory region is found around map position 127 000. (D) Repetitive signals (UEs) in an extended non-coding region. Note the apparent 2800 and 5000 periodicities of sharp destabilized sites in this range. Although all fragments (F20–F275) have a considerable affinity to the nuclear matrix (57–96% association in an equilibrium situation), competition experiments demonstrate that these UEs do not represent classical S/MARs [16].

 
While for a bacterial plasmid, superhelicity is the direct consequence of gyrase action, it also arises during the progression of tracking proteins such as DNA and RNA polymerases and—in eukaryotes—also due to the loss of nucleosomes (facilitated by histone hyperacetylation) [4]. Strand separation initiates at a nucleation centre (core-unpairing element, CUE) within the BUR, in case the underwound state is stabilized, either in the context of a circular DNA or of a chromatin domain that owes its existence to the bordering elements. These bordering elements, otherwise known as S/MARs (scaffold- or matrix attachment regions) are an essential attribute of higher order chromatin structures as they dissect the eukaryotic genome into independently regulated units (chromatin domains [5]). Each domain contains one gene together with its regulatory elements (upstream regulatory elements, enhancers, LCR), and accordingly the number of domains has been estimated (and partially verified) to be in the order of 30 000 with sizes that vary between about 4 kb (transcriptionally active) and 200 kb (rather inactive). Exceptions to this rule are clusters of co-regulated genes that may, with advantage, populate a single domain and larger arrays of transcriptionally inactive genes, which are not in contact with the transcription and replication machineries that are components of the nuclear matrix (i.e. the protein-/hnRNA-backbone of the nucleus). By necessity, these refinements implicate a discrimination of S/MARs into either constitutive or facultative variants. For the constitutive group, DNA–nuclear matrix contacts exist irrespective of cell type and transcriptional activity. Members of this family are mostly associated with a constitutive DNAse I hypersensitive site [6] and these are also the very sites at which domain-size fragments are generated early in apoptosis [7]. Facultative S/MARs on the other hand are elements, which need active transcription (presence of transcription factors, superhelicity or both) in order to associate with the matrix [8]. These contacts are usually marked by an activity-dependent or even cell-type specific DNAse I hypersensitive site.

In vivo DNA superhelicity is closely regulated, and it can induce the formation of locally unpaired regions that are involved in the initiation of replication and transcription [9]. The BURs not only form barriers between independently regulated domains [5, 10], but also support transcriptional initiation in a process that has been termed augmentation [11]. Proteins involved in constitutive contacts and their cooperation with regulatory factors that require locally denatured DNA for their binding have been described [3, 12, 13].

Definition of chromatin domain: type I interferon paradigm
The human type 1 interferon (IFN) gene cluster consists of 26 IFN genes and pseudogenes, distributed over 400 kb at Giemsa band 9p22. While IFNB1 forms an extended domain with many sites of regulatory potential, which together may constitute an LCR (locus-control region), the other interferon genes occur in three subclusters (I–III; Figure 1A). As a whole, the locus has gained attention because deletions that initiate here and include the adjacent tumour suppressor gene(s) are related to some of the most common genetic abnormalities that occur in numerous forms of cancer [7]. The S/MARs have been implicated in the mechanism of these deletions as they demarcate fragile sites in the genome (see the site marked ‘A172’ and ‘A1235’ in Figure 1A).

The first studies performed in this laboratory examined the organization of the 14 kb region containing the human IFN-ß (IFNB1) gene domain at the telomeric end of the 400 kb locus. The IFNB1 transcription unit is bounded by two extended, constitutive S/MARs of 7 kb (upstream) and 5 kb (downstream; Figure 1B'). An additional facultative S/MAR-element has been demonstrated between the gene and the downstream domain border. Owing to its favourable properties, the high affinity core of the upstream element (Figure 1B'') has found multiple uses for the design of novel vector types [14, 15] for which it provides the following properties:

  1. it insulates integrating vectors from the mostly negative influences of the surrounding (hetero-) chromatin [16];
  2. it provides a domain-opening function as it mediates the acetylation of histones [11];
  3. it provides long-term stabilization of gene expression because it prevents methylation dependent inactivation processes as they occur on Lys-9 of histone H3 and at CpG tracts in the DNA;
  4. as a consequence of their strand separation potential S/MARs are recombinogenic elements promoting recombination processes that may be desired for certain systematic modifications of target cells (homologous and site-specific recombination protocols [17]).

These activities motivated a detailed investigation of this and related elements by the combination of biomathematical and in vitro approaches. Figure 1B'' illustrates a distinct architecture across the BUR, which is composed of a regular succession of minima (unpairing elements, UEs) within the SIDD profile. This architecture is responsible for the association of prominent proteins of the nuclear matrix most notably SAF-A (scaffold-attachment factor A, otherwise known as hnRNP-U). Its parameters (over-all destabilization, extension and spacing of the UEs) have recently been amalgamated into a model that, for the first time, permits the precise prediction of interaction strength with the nuclear matrix and thereby its biological activity [3]. It should be noted that the over-all destabilization of an insert can be quantified in the context of a plasmid using the mentioned ampicillin-gene associated UEs as an internal standard (competition principle [3], Figure 1B'').

In contrast to the domain borders, the facultative S/MAR-region of IFNB1 consists of three strongly destabilized, widely separated elements at positions –500, –2000 and –3000 bp relative to the transcriptional start site, which could be correlated with regions of DNAse I hypersensitivity (HS3, HS2, HS1 in the above order, Figure 1B' and [12]). Electrophoretic mobility shift analyses demonstrated that either the known transcription factor YY1 or its recently discovered analogue YY2 [18] can be accommodated by an aaATGGt motif, which, within 5kb of upstream sequence, only exists twice at the flanks of the HS1 and HS2-associated peaks [12]. Both of these sites have to be occupied simultaneously to enable activation by two molecules of YY2, as these recruit a histone-acetyltransferse (GCN5) to the enhanceosome. It should be noted that, until recently, the enhanceosome-binding sequences upstream from position –110 constituted the only known control element that was implicated in the control of the IFN-ß genes in humans and in mice. Based on the present data, a refined mechanism for gene induction was proposed in which the ubiquitous factor YY1 provides inactivity by its competition with YY2 [13].

In spite of the apparent space limitation within the gene subclusters II and III, all functional gene members appear to be separated by efficient, though restricted S/MARs as well (Figure 1A). Figure 1C covers two functional IFN-alpha genes A10 and A7 in addition to a pseudogene (WP18). Interestingly, in this and several other cases the pseudogenes have lost flanking destabilized regions indicating that there is a selective pressure acting on the physicochemical properties investigated here [19].

In an extended non-coding region between the IFNB1- and IFNW1-genes, we detected a striking periodicity of rather restricted SIDD minima, which seem to obey a periodicity of roughly 2500 bp (Figure 1D). These elements had escaped prior in vitro S/MAR mapping efforts. Only their precise localization in the SIDD profile enabled a subsequent investigation, which led to the conclusion that the signals correspond to a new class of S/MARs with transcriptional augmentation- but no insulation-potential [16]. Initially, it has been suggested that this register of elements might be involved in levels of chromatin organization above the periodicity of DNA bend sites, which occur once per four nucleosomes [8]. Regarding the increasing number of reports dealing with TUFs (transcripts of unknown function) that are presently discovered across large sections of the human genome, this region may alternatively serve yet unknown regulatory functions (report by T. Gingeras at the 2005 BITS meeting and [20]). Also, in this case the SIDD concept would have opened the door for novel insights into DNA structure–function relationships.

Intronic S/MARs
The example of a facultative element has already shown that S/MARs cannot simply be considered to be static delimiters of functional domains. This view is supported by the detection of S/MARs within the first intron of a number of genes, where they have regulatory potential [21]. Such a relationship has recently also been confirmed in a whole-genome screen on Arabidopsis [22] (communicated by T. Werner at the 2005 BITS meeting; Tetko et al. 2005, submitted).

Classical examples of this category are S/MARs that are associated with the immunoglobulin {kappa}- and µ-chain genes [23, 24]. Since, by definition, intronic S/MARs are transcribed, and since they do not impede passage of RNA polymerase II, their occupation must be regulated. Ig-µ genes are formed by the joining of three gene segments that are separated in the germline, i.e. the variable (V-), diversity (D-) and joining (J-) regions. Two segments (V and J) are linked to generate {kappa} and {lambda} light chain genes [25]. Key to this control is the regulated access of the lymphoid-specific RAG-recombinase proteins to the recombining loci. The process commences at the µ-chain locus at the pre-B I stage, followed by rearrangement at the {kappa}-locus. Finally, the {lambda}-locus is rearranged, unless the expression of a functional {kappa}-chain has ablated this process. Prior to rearrangements, germline transcription is observed, which serves to create accessibility as it is associated with changes in the methylation status and over-all chromatin structure.

For the immunoglobulin µ- and {kappa}-genes, transcription before and after rearrangement depends on an enhancer within one of the introns. Experiments with transgenic mice have shown that promoter activity also requires the S/MARs at one ({kappa}) or both (µ) flanks of the enhancer. While the µ-enhancer alone permits access to its vicinity, accessibility is significantly extended by the S/MAR(s) in a process that correlates with extended demethylation [26].

The juxtaposition of S/MARs with transcriptional enhancer elements has been evolutionarily conserved within the Ig{kappa} genes of the mouse, rabbit and human. Mouse {kappa}-constructs lacking the S/MAR have a lower and erratic expression in transgenic animals and, particularly, in cultured cells. Just as its µ-counterpart, the {kappa}-gene becomes demethylated during B-cell maturation. While any S/MAR sequence is sufficient for this reaction, tissue specificity is mediated by NF-{kappa}B binding sequences within the {kappa}-enhancer [27]. Deletion of the Ig{kappa}-intronic S/MAR results in hyperrecombination of closeby {kappa}-genes as well as to a decreased level of somatic hypermutation [28]. These observations suggest that its sequences contribute to changes in chromatin structure.

In mice, {lambda}-chains form only about 5% of the total serum immunoglobulin light chains and they are much less heterogenous than {kappa}-chains. Although no S/MAR has been found in the J–C introns of the {lambda} clusters, the SIDD approach led to the detection of an exceptionally strong element in the C{lambda}2-J{lambda}4 and C{lambda}1-J{lambda}1 introns [29]. In contrast to the µ- and {kappa}-genes, where the loops are anchored at the same site before and after rearrangement due to the persistence of the S/MAR between the J- and C-fragments, the rearrangement at the {lambda} loci leads to the loss of the respective introns (Figure 2). The elements upstream from {lambda}1 and {lambda}4 might therefore be involved in the long-distance interaction during activation or in the rearrangement process itself and their function may later be taken over by destabilized sites that arise downstream from C{lambda}1 (Figure 2, lower part). To what extent these differences between the light-chain loci contribute to the apparent dominance of the {kappa}-chains remains to be determined.


Figure 2
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  		Figure 2: An S/MAR in the un-rearranged Ig{lambda} light-chain locus. The S/MAR character of a destabilized site in the C{lambda}3-J{lambda}1 (map position 4700) and the analogous C{lambda}2-{psi}J{lambda}1 region (not shown) was verified by wet-lab techniques [29]. In contrast to the situation for the µ- and {kappa}-chain genes the element is situated at a position that is lost during the rearrangement process. Its function must thereby be restricted to germline transcription, where it may set the stage for rearrangement. New dominant sites appear after this process (lower part).

 
S/MARs by design
Small circular episomes of DNA viruses such as SV40, BPV and EBV are well-studied minimal models of a chromatin domain the more, as their function and maintenance has been associated with the nuclear matrix [9]. The practical application of these vehicles is limited, however, by the fact that their function depends on virally encoded, oncogenic factors. The design and verification of a non-viral circular episome that is in the position to recruit components from the replication apparatus of the host cell via a S/MAR came therefore as a breakthrough [9]. Work in the subsequent years clearly showed that there is an inverse correlation between an episome's molecular size (4–12 kb) and its extrachromosomal maintenance. These observations triggered various efforts to minimize these vehicles by a rigorous definition of their molecular components.

An early approach in this direction concerned the replacement of the original 2.0 kb S/MAR segment from the IFNB1-upstream domain border by a minimal element that had been obtained by oligomerizing an UE (bottom parts of Figure 3). Binding parameters and transcriptional activities of various oligomers have been determined and the 620 bp tetrameric insert that had been derived from the 155 bp CUE was found to maintain episomal replication [30] (details under http://www.pnas.org/cgi/content/full/0401355101/DC1/8).


Figure 3
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  		Figure 3: Replication of non-viral episomes depends on a S/MAR element. While the original vector contains the S/MAR element (‘SAR-E’) shown in Figure 1B'', a minimal version, which provides full replication capacity was obtained when this element was replaced by an artificial tetramer containing four identical copies of the CUE [30]. Current evidence shows that only the eukaryotic elements of the vector (bottom part of the box) are required for replication as an episome.

 
It is particularly this series of experiments, which demonstrates the utility of the SIDD algorithm for a deeper molecular understanding of the molecular functions of S/MAR modules and their combinations. The door is therefore open for the rational design of autonomous chromatin domains, which, in the extreme case, are circular, non-viral episomes with a single domain boundary (S/MAR). Current evidence suggests that such systems will efficiently circumvent the drawbacks that are otherwise due to suppression mechanisms acting on transgenes.


   PERSPECTIVE
TOP
 ABSTRACT
 INTRODUCTION
 PERSPECTIVE
References
 
It appears that distinct patterns of UEs are associated with major aspects of both replication and transcription. Our work indicates that the SIDD method may supplement or even partially replace current efforts such as that reported by Crawford et al. [31] to identify the location of all cis-acting regulatory elements by a genome-wide recovery of DNAse I hypersensitive sites. Monitoring the ‘stress-induced duplex destabilization’ (SIDD) therefore qualifies as a method to indicate the sites where ‘strain initiates domain decondensation’ (SIDD).


Key Points

  • Gene regulation is not only explained by the sequence but also by the higher structure of DNA elements.
  • An important prerequisite is the strand separation potential under superhelical strain (SIDD).
  • Independently regulated chromatin domains can be localized in an SIDD profile together with the regulatory elements (DNAse I hypersensitive sites) they contain.
  • We apply these principles to demonstrate a remote-control mechanism for the human interferon-beta gene, to localize fragile genomic sites and to unravel the prerequisites for the rearrangement of immunoglobulin genes.
  • Modular S/MAR-elements with predictable properties can be designed based on their SIDD properties.

 


   FOOTNOTES
 
Silke Winkelmann is a PhD student in the group of JBO where she develops novel methods to study transcriptional properties as they relate to nuclear structure.

Martin Klar received his PhD and a subsequent prize in 2005 for his work in the same group dealing with the remote control of the human and mouse interferon-beta genes. He could verify the major predictions of SIDD profiles. http://opus.tu-bs.de/opus/volltexte/2005/713/pdf/DissMK.pdf

Craig Benham is the senior biomathematical counterpart of the group. He has an AB degree from Swartmore College and a PhD in Mathematics from Princeton University. His studies of biopolymer structure started when he worked with John Kozak at the University structure (proteins) and during his postdoctoral years with Max Delbrueck at Cal Tech. After leading the Department of Biomathematics at Mount Sinai, NY, he became a founding Associate Director of the UC Davis Genome Center. Contacts to the Braunschweig group date back to 1994, the first joint publication to 1997.

Prashanth AK joined Craig Benham at Mount Sinai and now he shares his research programmes at Davis. Nowadays he is the active cooperation partner of the Braunschweig people.

Sandra Goetze did her PhD in the lab of Prof. Bode (1998–2001) working on the identification and biochemical analysis of boundary elements under consideration of biomathematical models. Currently she is investigating the three-dimensional chromatin organization in the human interphase nucleus in the context of an FP6 European Program in the lab of Prof. van Driel in Amsterdam. http://opus.tu-bs.de/opus/volltexte/2004/587/pdf/Dissertation.pdf

Angela Gluch (born Knopp) initiated participation in the human genome project together with JBO. The major concepts realized in the present contribution go back to her initiative. She received PhD in 2001. She may return to the group after maternity leave in 2006. http://opus.tu-bs.de/opus/volltexte/2001/200/pdf/Dissertati.pdf

Juergen Bode studied Organic Chemistry at the Technical University of Braunschweig. His switch to Biochemistry occurred during postdoctoral years at the California Institute of Technology (CalTech; enzyme and neuroreceptor structures with Michael A. Raftery) and University of Oregon, Eugene (UofO; enzyme kinetics with Sidney A. Bernhard). The final direction of research initiated in the year the nucleosome was discovered. It widened to aspects of nuclear structure as these relate to gene expression. He is a group leader (Epigenetics) at GBF (German Research Center of Biotechnology at Braunschweig) and a Professor of Biochemistry at the Technical University (http://juergenbode.de.vu; http://cvjbo.de.vu).


   References
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 INTRODUCTION
 PERSPECTIVE
 References
 

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