Histone Modifications Influence the Action of Snf2 Family Remodelling Enzymes by Different Mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.
Introduction
Packaging DNA into nucleosomes and higher-order chromatin structures restricts access to the underlying genetic material. Thus, the manipulation of chromatin is a key step in many nuclear processes such as transcription, replication and repair. One of the means by which eukaryotes achieve this is through the use of ATP-dependent chromatin remodelling enzymes that non-covalently alter its structure. These enzymes are members of a diverse group of proteins named after the archetypal Saccharomyces cerevisiae Snf2 protein; the Snf2 family. Multiple members of this family of proteins are present in the sequenced genomes of eukaryotes of which the chromatin remodelling enzymes form distinct sub-groupings.1 Recent crystal structures of the catalytic domain of two Snf2 related proteins2,3 highlight structural similarities with the RecA domain found in a broad range of helicase related proteins. Snf2 proteins use the energy from ATP hydrolysis to alter DNA–protein interactions. However, unlike bona fide helicases, the action of chromatin remodelling enzymes is not generally associated with separation of DNA strands. Instead they act to catalyse dynamic transitions in chromatin structure. This can result in a variety of different outcomes. In vitro, chromatin remodelling enzymes have been shown to cause alterations to DNA accessibility, effects on DNA and chromatin topology, partial or complete removal or exchange of histones from nucleosomes and translational changes in the position of the intact histone octamer along DNA.4
How these proteins interact with chromatin and the mechanism by which chromatin remodelling is achieved remains unclear. However, in many cases it is apparent that histone tail domains are important regulators of chromatin remodelling. Genetically, there are links between histone tails and remodelling. Deletion of the H2B tail bypasses the need for the SWI/SNF complex in the regulation of several genes,5 and deletion of the H2A tail affects transcription of some SWI/SNF dependent genes.6 Importantly, post-translational modifications of histone tails affect chromatin remodelling. The recruitment and activity of the SWI/SNF complex in vivo is widely linked to that of the SAGA histone acetyltransferase complex.7–9 In addition, many chromatin remodelling complexes contain domains shown to bind modified amino acids, such as bromo- and chromodomains, PHD and WD40 domains.10 In these cases modifications have been proposed to act as epitopes that facilitate recruitment of proteins containing the appropriate recognition domains. For example, histone acetylation has been found to facilitate retention of SWI/SNF on nucleosome arrays11 in a way that is dependent on bromodomains within the Snf2 protein.12
The majority of the best characterised modifications occur within the non-globular histone tail domains which protrude from the core of the nucleosome. These histone tails constitute up to 30% by mass of histones, but are not visible in the crystal structures of nucleosomes due to their high intrinsic flexibility and have been thought to be largely unstructured.13 Their importance is highlighted by the fact that deletion of histone tails has wide spread effects on chromatin structure and gene regulation.14–16 Histone modifications have been correlated with a variety of chromatin states. On a genome-wide basis, histone H3 K4 tri-methylation and H3 acetylation are strongly correlated with active transcriptional start sites, phosphorylated H2A.X foci mark sites of DNA damage, and H3 K9 tri-methylation promotes the formation of heterochromatin via interaction with HP1.10,17 This suggests that histone post-translational modifications are an important means by which cells spatially and temporally regulate specific loci independently of bulk chromatin.
Rigorously studying the effect of histone tail modifications in vitro has been hampered by the difficulty in isolating histones with defined and homogeneous modifications. To circumvent this we have adopted an approach to chemically synthesise modified histones by means of native chemical ligation. This technology has previously been applied to generating chromatin with uniform histone N-terminal tail modification patterns.18–20 Using this approach we show that histone modifications can affect nucleosome remodelling via distinct pathways. Tetra-acetylation of histone H3 results in a modest increase in the rate of intrinsic nucleosome repositioning. The ATP-dependent remodelling enzyme RSC shows a striking preference for H3 but not H4 tetra-acetylated chromatin, remodelling this 16-fold faster than the control. By measuring the kinetic parameters of the remodelling reaction with a real-time ATPase assay, we show that this is due to a lower Km value for H3 acetylated nucleosomes. In contrast, tetra-acetylation of histone H4 affects nucleosome remodelling by the Isw2 enzyme via altering the Kcat rather than the Km of the ATPase reaction. We also show that the yeast Chd1 enzyme is dependent on the H4 tail for efficient nucleosome remodelling in a manner similar to Isw2. Remarkably, histone H4 tetra-acetylation affects the rate of RSC catalysed nucleosome transfer in trans but not nucleosome sliding in cis. Thus, nucleosome modifications can affect enzyme catalysed remodelling reactions by altering the outcome of the reaction, through allosteric effects on ATPase activity and by acting as binding epitopes for recruitment.
Results
Generation of modified chromatin
Modified histones were generated by ligating a bacterially expressed Xenopus laevis globular histone containing an N-terminal cysteine and a tail peptide with the modification of choice synthesised chemically as a C-terminal thioester. The ligation was based on the protocol described by Dawson & Kent21,22 and used thiophenol as the catalyst (Figure 1(a)). A key requirement of the globular histone is that it has an uncapped N-terminal cysteine available for ligation. One strategy that has been used previously to ensure that the initiator methionine is removed is the introduction of a protease cleavage site just before the cysteine to expose it post-translationally.20 Mass spectrometry (Supplementary Data, Figure 1) and the work of other laboratories19,23 show that removal of the initiator methionine in vivo by Escherichia coli methionine aminopeptidases is efficient when cysteine is the adjacent amino acid. This alleviates the requirement for more complex systems for generation of N-terminal cysteine residues.
Polyacrylamide gel electrophoresis reveals that under the conditions used, ligation does not proceed to completion (Figure 1(b), lane 1). However, the full-length modified histone can be separated from the unreacted globular domain and tail peptide via multiple rounds of ion exchange chromatography to obtain material that was greater than 95% pure (Figure 1(b)). The overall efficiency of ligation and subsequent purification is approximately 20% (data not shown). When modified histones were combined with other core histones to form histone octamers and purified by size exclusion chromatography, they displayed identical elution profiles to unmodified octamers (data not shown). Modified histone octamers could be reconstituted onto DNA fragments to form nucleosomes. Interestingly, acetylation of H3 and to a lesser extent H4 reduced the migration of nucleosomes on native polyacrylamide gels (Figure 1(c)). This has been observed previously in the Bradbury laboratory using hyperacetylated histones purified from HeLa cells24 and suggests that linker DNA conformation may be affected. This altered electrophoretic migration is not due to the ligation procedure as nucleosomes containing methylated lysine residues did not show this behaviour (not shown).
Lysine acetylation can alter intrinsic nucleosome mobility
Although nucleosomes are stable with respect to dissociation, they can undergo a range of dynamic fluctuations in their structure. For example, following thermal incubation nucleosomes are frequently observed to redistribute to thermodynamically favoured locations.25 This movement of nucleosomes from one translational position on DNA to another can be followed using native PAGE.26 Acetylated and control nucleosomes were reconstituted onto differentially labelled fluorescent DNA, mixed, incubated thermally and run on a native gel (Figure 2(a)). The different nucleosomes can then be visualised separately using selective excitation and emission filters. From this the proportion of nucleosomes at the initial and final position can be measured at each time point. The rate at which nucleosomes accumulate at their destination can then be plotted and a hyperbolic curve fitted to the data enabling the initial rate of redistribution to be estimated (Figure 2(b)–(e)). This approach has proved more reliable at directly comparing the behaviour of different nucleosomes.27
Although native thiol ligation results in a normal peptide bond without the introduction of unusual chemical moieties, it requires the presence of a cysteine residue at the point of ligation. For this reason, the unmodified control nucleosomes used as a reference are point mutants containing a cysteine at the equivalent position to the modified histones. The sites selected for ligation involved substitution of cysteine for amino acids with similar overall dimensions (e.g serine to cysteine). None the less it was important to show that introduction of these mutations did not affect the behaviour of nucleosomes. Supplementary Data, Figure 2 shows a comparison of the thermal mobility of wild-type and cystein point mutant nucleosomes showing that the cystein mutations themselves have no discernable effect. The effects of the cysteine point mutations were also compared to wild-type histone sequences in other assays described below and in all cases no significant effect was detected (Supplementary Data, Figures 2, 3, 4; and data not shown). As a histone bearing the relevant cysteine point mutation is most closely matched to the product of a peptide ligation reaction, we have generally presented data using the point mutant as a control rather than the wild-type histone sequence. However, we are not aware of any cases where the choice of control affects the conclusions drawn.
H4 acetylated nucleosomes were observed to be repositioned at the same rate as control nucleosomes (Figure 2(b)). In contrast, nucleosomes containing acetylated H3 relocated twice as fast as the control (Figure 2(c) and (e)). To test to what extent this was due to the partial neutralisation of basic charge that occurs upon acetylation of lysine residues, a histone H3 construct with lysine to alanine mutations at the four acetylated positions (H3 K9, 14, 18, 23A) was compared to wild-type (Figure 2(d) and (e)). This nucleosome repositioned at the same rate as the control indicating that charge neutralisation is not the sole mechanism by which acetylation affects nucleosome mobility.
The RSC complex preferentially repositions tetra-acetylated H3 but not H4 nucleosomes
RSC is an abundant and essential yeast chromatin remodelling complex that is closely related to SWI/SNF.28 A notable feature of the RSC complex is that it contains almost half of the known bromodomains in the S. cerevisiae genome. As bromodomains within other proteins have been found to recognise specific patterns of histone acetylation,29,30 a distinct possibility is that histone acetylation will influence the action of the RSC complex. To test this, the ability of RSC to remodel either H3 or H4 acetylated chromatin relative to unmodified control chromatin was assayed. RSC showed a dramatic preference for H3 (K9, 14, 18, 23) tetra-acetylated chromatin compared to an unmodified control (Figure 3(a)). From the initial rates of remodelling derived from the plotted data, this was calculated to be 16(±1.5)-fold faster than control (Figure 3(d)). When the rate of H4 (K5, 8, 12, 16) tetra-acetylated nucleosomes was measured, this was found to be indistinguishable from the rate of an unmodified control (Figure 3(b) and (d)).
It is also worth noting that although the rate at which the H3S28C control nucleosomes are shifted in Figure 3(a) is slower than the rate at which the H4V21C nucleosomes are shifted in Figure 3(b), this should not be interpreted as indicating that the S28C mutation reduces the rate at which nucleosomes are repositioned by RSC. The reason that S28C nucleosomes are moved slower is due to the preferential engagement of RSC with acetylated nucleosomes at the expense of non-acetylated nucleosomes in the reaction mixture, as they are essentially in competition with each other. Indeed, comparing the rate of acetylated nucleosomes to either wild-type or cysteine containing control nucleosomes shows the same effect (Supplementary Data, Figure 3) This illustrates the importance of only making direct comparisons between the two templates present in the same remodelling assay. In this case, our data show that H3 acetylation stimulates nucleosome slding whereas H4 acetylation does not. Thus, none of the seven bromodomains found within the RSC complex appear to interact with acetylated lysines within the H4 tail in a way that affects nucleosome sliding. This is consistent with the observation that the relative rate of remodelling of a H3 and H4 acetylated nucleosome is not faster than a nucleosome that is just acetylated on the H3 tail (Figure 3(c) and (d)).
H3 tetra-acetylation influences remodelling by the RSC complex by affecting Km but not Kcat of ATP hydrolysis and acts largely via H3 K14 acetylation
The increased rate with which RSC repositions H3 acetylated nucleosome could be due to the modified lysine residues acting to recruit RSC or by allosterically affecting the remodelling reaction. To differentiate between these two options, the kinetic parameters of the ATP hydrolysis reaction were measured using a real-time fluorescent ATPase assay.31 The assay hinges on using a phosphate binding protein (PBP) labelled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), as a sensor for the amount of inorganic phosphate (Pi). On binding Pi, the labelled protein (MDCC-PBP) undergoes a shift in its emission wavelength coupled with a fivefold increase in fluorescence. When performed in a fluorimeter, this assay has the advantage of measuring ATP hydrolysis in real-time, from which kinetic parameters such as Km and Vmax are determined by non-linear fitting to the Michaelis–Menton equation (Figure 4(a)–(d)). We find that RSC has approximately threefold lower Km (tighter binding) for H3 acetylated nucleosome compared to the unmodified control, without affecting the Kcat of ATP hydrolysis (Figure 4(e)). This is consistent with preferential recruitment of RSC to H3 acetylated chromatin and the preferential binding of RSC to H3 acetylated nucleosomes (Supplementary Data, Figure 5). In contrast, RSC does not preferentially bind H4 acetylated nucleosomes, as the Km for these is the same as for the unmodified control (Figure 4(e)). Consistent with this, the Km value of the doubly H3, H4 acetylated nucleosome is indistinguishable from that of the H3 acetylated nucleosome (Figure 4(e)).
Previous work from the Cairns laboratory has suggested the tandem bromodomains of the RSC4p subunit of the RSC complex interact with acetylated H3 K14.32 To test whether the single acetylation of lysine 14 is responsible for the dramatic effect of H3 (K9,14,18,23) tetra-acetylation on RSC activity, the singly acetylated nucleosome was generated and put through the same ATPase assay described above. From Figure 4(e) it can be seen that as for H3 tetra-acetylated nucleosomes, the Kcat value remains unchanged. In contrast, the Km value was significantly lowered, almost to the levels of the tetra-acetylated construct, suggesting that acetylation at a single residue, K14, confers the majority of the effect observed upon tetra-acetylation of the H3 tail.
The H4 tail and its acetylation influence remodelling by Isw2 by affecting the Kcat of ATP hydrolysis
Subfamilies of Snf2 family chromatin remodelling proteins contain different motifs capable of interacting with histones raising the possibility that they may be regulated in different ways by histone modifications. One of the defining characteristics of the Iswi subfamily of remodelling enzymes is that they require a particular epitope within the H4 tail for efficient remodelling activity.33–36 One of the lysine residues adjacent to this motif, H4 K16, can be acetylated, and there is evidence that this reduces the activity of ISWI containing remodelling complexes.19,37 When we compared the relative rate of mobilisation of H4 tetra-acetylated nucleosomes relative to an unmodified control by the yeast Isw2 enzyme, we found that the rate is approximately 1.5-fold slower (Figure 5(b) and (c)). This reduction, whilst significant, is not as severe as deletion of the H4 tail (Figure 5(a) and (c)). However, its biological importance is underlined in studies on flies, where defects in the genetic interaction between H4 K16 acetylation and ISWI result in large scale chromosomal abnormalities.37
Applying the same kinetic analysis performed previously to RSC shows that deletion or acetylation of the H4 tail lowers the turnover rate of the hydrolysis reaction (Kcat) without affecting nucleosome binding (Km) (Figure 5(d)). We have confirmed this by performing gel shifts of either full length or gH4 nucleosomes with Isw2 and observe no difference in binding (Supplementary Data, Figure 6). This contrasts with the effect of histone acetylation on remodelling by the RSC complex and shows that histone modifications can affect chromatin-remodelling enzymes at different stages of the reaction cycle.
The yeast Chd1 enzyme requires the H4 tail for efficient chromatin remodelling
Chd1 belongs to a phylogenetic subfamily that is distinct from either of the previously studied enzymes1 and contains a chromodomain that has the potential to recognise histone modifications. To investigate the histone dependence of this enzyme, we first tested the effect of deleting individual histone tails. We found that Chd1 is unable to efficiently slide nucleosomes lacking the H4 amino terminal tail (approximately sixfold slower than control: Figure 6(a), compare lanes 1–6 with 7–12). This requirement for the H4 tail is similar to that seen with Iswi remodellers such as Isw2, although the magnitude of the effect is lower. Mutation of residues 16–19 to alanine caused a 4.4-fold reduction in Chd1 activity (Figure 6(b)). Although this effect is significant it does not fully match the effect of a H4 truncation raising the possibility that the amino acids recognised by Chd1 include residues in addition to those within the 16–19 region.
Acetylation of residues within the H4 tail also affected Chd1's nucleosome remodelling activity. Indeed, when this was tested, there was a modest 1.4-fold reduction in the initial rate of remodelling of acetylated H4 versus unmodified nucleosomes (Figure 6(c) and (d)). This raised the question of whether, as in the case of Isw2, the H4 tail was an allosteric activator of ATP hydrolysis or whether it was required as a binding epitope to recruit Chd1 to nucleosomes. Comparing the ATP hydrolysis rates of Chd1 with full-length and gH4 nucleosomes revealed that binding (Km) was unaffected (see also Supplementary Data, Figure 6(b)) and it was again the catalytic turnover rate which decreased in the absence of the H4 tail (Figure 6(e)).
H4 acetylation leads to loss of histones in trans
Studies with the RSC and SWI/SNF complexes in vitro have shown that in addition to being able to redistribute nucleosomes along DNA fragments, they can transfer histone octamers from one DNA fragment to another at lower efficiency. This entails the displacement of the histone octamer from one molecule of DNA and subsequent transfer onto a separate one. The SWI/SNF complex has been implicated in the disassembly and subsequent reassembly of nucleosomes at the Pho5 promoter.38,39 This loss of nucleosomes at the promoter is correlated with spikes in histone acetylation levels, leading to the hypothesis that ATP-dependent remodellers may recognise this signal and promote transfer. To test whether there is a direct link between histone modification and octamer transfer, the efficiency of octamer transfer from different chromatin substrates by the RSC complex was measured.
The octamer transfer reaction involves incubating a “cold” donor nucleosome with a “hot” 32P-labelled accepter DNA fragment in the presence of RSC and ATP. The reaction products were run on a native polyacrylamide gel and transfer is indicated by a shift in mobility of the “hot” accepter DNA. If the accepter DNA has a histone octamer transferred onto it, it should now have the same mobility as a control nucleosome reconstituted onto the same fragment (Figure 7(a)).
This was quantified as the fraction of radiolabelled free DNA incorporated into nucleosomes, as measured by mobility on a native polyacrylamide gel (Figure 7(b) and (c)). The amount of octamer transfer catalysed increased over time and was dependent on both ATP (Figure 7(b), lanes 1–3) and RSC (data not shown). As expected, octamer transfer from H3 acetylated nucleosomes was greater compared to unmodified nucleosomes (Figure 7(b), compare lanes 1–3 and 4–6). This is consistent with RSC having a lower Km for H3 acetylated nucleosomes (Figure 4) and the preferential nucleosome sliding seen in Figure 3. Surprisingly, when we looked at H4 acetylated donor nucleosomes, these were transferred considerably more efficiently than unmodified nucleosomes. Indeed, the amount of transfer was almost as high as that of H3 acetylated nucleosomes (Figure 7(c)), despite RSC not showing any increased affinity towards H4 acetylated chromatin (Figure 4). A doubly H3 and H4 acetylated nucleosome was in turn transferred more efficiently than either of the single histone acetylations. This suggests that H4 acetylation does not act at the level of stimulating RSC directly but rather by predisposing the nucleosome to be remodelled in trans, perhaps by destabilising it. Consistent with this, we find that H4 acetylated nucleosomes are more prone to thermally induced histone H2A/H2B dimer loss than H3 acetylated nucleosomes (Supplementary Data, Figure 7).
Discussion
Histone acetylation alters the intrinsic dynamic properties of nucleosomes
Since they were discovered in the 1960's histone modifications have been predicted to affect chromatin structure and gene regulation.40 Subsequent studies have revealed correlations between histone modifications and transcription. For example, the recent application of genome-wide microarrays to map histone modifications, reveals that modifications such as H3 acetylation are found at the promoters of active genes and that their levels correlate with that of transcription.41–46 There are two prevailing views as to how modifications such as acetylation affect transcription. The first is based on the recruitment of activators due to recognition via modification binding modules. The number of modifications and respective binding domains identified has resulted in the hypothesis that these may form a code, which is read to determine a specific response.47 Alternatively it has been suggested that lysine acetylation may have a structural effect on chromatin structure by neutralising the charge interactions between histone lysine residues and DNA, resulting in a more open conformation.48–50 Several lines of evidence suggest that this can destabilise chromatin fibres.19,51–53 Histone acetylation may also affect the structure of mononucleosomes. Several studies suggest that DNA within acetylated nucleosomes is more accessible.48,49,54 It has been shown that acetylated histone tails adopt a more α-helical conformation,55 and that acetylated nucleosomes have a reduced linking number,24 consistent with wrapping less DNA. However, in this view, the role of individual histone modifications has been less clear. Here we show that acetylation of the H3 tail increases the inherent thermal mobility of nucleosomes whereas acetylation of H4 does not (Figure 2(e)). This increase in mobility does not appear to be due to charge neutralisation alone, as mutation of the relevant lysine residues to alanine did not produce the same effect (Figure 2(d)). The mechanisms behind this are not immediately clear; however, it is interesting to note that acetylation of H3 but not H4 increased the distance between the arms of linker DNA within a mononucleosome, as measured by FRET.53 We have also recently observed that individual histone tails have non-redundant effects on nucleosome dynamics,27 suggesting that the relationship between histone modification and chromatin dynamics may be more complex than initially appreciated. It will be interesting to examine how these effects interact with each other to alter chromatin structure and function in vivo.
Histone acetylation affects ATP-dependent chromatin remodelling through altering either the Km or Kcat of ATP hydrolysis
As their names suggest, ATP-dependent remodelling enzymes use the energy of ATP hydrolysis to alter chromatin structure. Based on the helicase domain of the catalytic subunit, chromatin remodelling complexes can be classified into a number of different subfamilies.1 Here we look at examples from three distinct subfamilies of chromatin remodellers: Chd1, Isw2 and RSC. The ATPase activity of these enzyme classes is stimulated by DNA and nucleosomes. Whereas it had been noted that histone tails and modifications affected their remodelling activity, it was not clear in many cases whether this was due to altered binding of modified nucleosomes or due to altered rates of ATP hydrolysis after binding. Using a real-time ATPase assay we show that depending on the particular enzyme involved either may be the case.
Analysis of the RSC complex confirmed that the improved remodelling of acetylated nucleosomes is due to higher substrate binding affinity and not due to increased ATP turnover (Figure 4(b)). This is consistent with previous observations of bromodomain containing remodelling enzymes, such as SWI/SNF, which show improved binding to acetylated chromatin templates.12,56 Remarkably, given the large number of bromodomains, the RSC complex shows no improved binding of H4 tetra-acetylated nucleosomes but rather, specifically H3 acetylated nucleosomes. By using chromatin with defined modification patterns we show that mono-acetylation at lysine 14 results in an increase in binding almost to the level seen with H3 tetra-acetylation. Consistent with this the Rsc4p subunit of RSC interacts genetically with H3 K14 but not H3 K9.32 Studies from the Workman laboratory have shown that SWI/SNF and RSC have increased affinity for nucleosomes that have been acetylated by the NuA4 HAT complex.12,56 We show here that RSC does not preferentially bind H4 K5,8,12,16 tetra-acetylated nucleosomes (Figure 4(b)); however, these observations can be reconciled by the fact that NuA4 shows HAT activity towards other histones besides H4, most notably H2A,57,58 and it may be this which results in the improved binding. For this reason, it would be interesting to test the affinity of RSC for nucleosomes containing acetylated H2A or H2B. Indeed, in high-resolution microarrays, H2A K7 acetylation but not H2B K16 acetylation is typically found in the same region of genes as H3 acetylation, and both are positively correlated with transcription.46
In the case of Isw2, we show that acetylation of H4 reduces nucleosome remodelling by lowering the Kcat of ATP hydrolysis and not by inhibiting nucleosome binding (Figure 5(c)). Thus, the unmodified H4 tail is required as an allosteric activator of ATP hydrolysis. This is consistent with the previous observation that acetylated H4 peptides have a reduced ability to stimulate ATP hydrolysis by dISWI in comparison to unmodified peptides.34 We also find that Chd1 activity is affected by modification and alteration to the H4 tail in a similar way to the Isw2 complex (Figure 6). It is notable that in the case of both Chd1 and Isw2, the effects of tail acetylation or truncation on ATPase activity are smaller than the effects on nucleosome sliding. It is possible that there is some amplification of the effect on ATPase activity due to multiple rounds of remodelling being required for repositioning. Alternatively, alterations to the H4 tail may reduce the efficiency with which these enzymes reposition nucleosomes in addition to altering their ability to hydrolyse ATP.
Previously, the requirement for the H4 tail for efficient nucleosome remodelling had only been observed for members of the Iswi subfamily of remodelling enzymes. However, there is evidence that there may be a significant degree of overlap in the function of Chd1 and Iswi subfamilies in S. cerevisiae. Isw1, Isw2 and Chd1 are all involved in transcription termination59 and their combined deletion is synthetic lethal with cellular stress.60 These complexes also reposition nucleosomes onto the same subset of thermodynamically favourable positions in vitro.61 Interestingly, like members of the Iswi subfamily, Chd1 is efficient in the generation of regularly spaced chromatin arrays,62 suggesting that a tightly coupled functional interaction with the H4 tail may be important for this activity. Studies with the similar Drosophila Mi-2 chromatin remodelling complex have shown that while its ATPase activity is stimulated by the assembly of histones into nucleosomes, this does not require histone tails.63 Whereas Mi-2 has similarity to Chd1, it is a member of a phylogenetically distinct group,1 suggesting that of the CHD-like remodellers, dependence on the H4 tail may be restricted to the Chd1 subfamily.
H4 acetylation promotes octamer transfer in trans by the RSC complex
A distinct activity of the SWI/SNF and RSC complexes is their ability to remove nucleosomes and displace histone octamers in trans. The targeted removal of nucleosomes has been shown to be an important phenomenon in nuclear processes such as transcription64–66 and DNA repair67,68 and interestingly chromatin remodelling enzymes are involved in both.
In testing the role of histone modifications on octamer transfer in trans by RSC, we found that H3 tetra-acetylation resulted in a large increase in transfer (Figure 7(b) and (c)). This effect of H3 acetylation in promoting octamer transfer can be explained through improved recruitment due to the increased affinity for the modified nucleosome. Indeed, octamer transfer by the homologous SWI/SNF complex has been shown to require its bromodomains to mediate octamer transfer of SAGA acetylated nucleosomes.69 An unexpected finding was that H4 tetra-acetylation also had a stimulatory effect (Figure 7(b) and (c)), in stark contrast to the results seen with nucleosome repositioning in cis (Figure 3(b) and (d)). However, the additional effect of H4 acetylation was confirmed by the fact that a doubly H3 and H4 acetylated nucleosome was transferred better still than a singly H3 acetylated nucleosome. Kinetic analysis of ATPase activity clearly shows that H4 tetra-acetylated nucleosomes are not bound any better than unmodified nucleosomes and they do not alter the rate of ATP hydrolysis by RSC (Figure 4(b)). Thus, the increase in octamer transfer seen with H4 acetylated nucleosomes is not due to the modification recruiting or stimulating RSC activity but rather by affecting the nucleosome in such a way as to make it easier to transfer. In agreement with this, we find that H4 acetylated nucleosomes show increased thermally induced H2A/H2B dimer loss (Supplementary Data, Figure 7), indicating that this modification can affect chromatin dynamics independently of external factors. Interestingly, previous studies have shown that acetylation reduces the thermal stability of nucleosomes and H4 tetra-acetylation in particular reduces the thermal stability of nucleosomes almost to the level of bulk acetylation.70,71 The finding that histone acetylation improves the efficiency of histone octamer transfer by remodelling enzymes helps to explain discrepancies between previous studies of transfer efficiency. Previous studies that used native histones bearing modifications observed octamer transfer at a higher efficiency than was detected in another study using recombinant unmodified histones.72
The idea that acetylation of different histone tails may not be functionally equivalent is not a new one. Genome-wide microarray studies in yeast have shown that not all acetylation is positively correlated with transcription,42,46 and H4 acetylation does not substitute for H3 acetylation at Adr1-dependent genes.73 We propose that histone modifications may provide a means to facilitate histone eviction by ATP-dependent chromatin remodelling enzymes. Thus, acetylation, particularly of H4, may act in parallel with recruitment by activation domains to promote the removal of nucleosomes by remodelling enzymes such as RSC and SWI/SNF.74 This may also have relevance to processes other than transcription such as DNA repair. Intriguingly, H4 acetylation is very rapidly targeted to these regions75,76 making it tempting to speculate that this modification also acts to promote nucleosome loss during DNA repair.
Experimental Procedures
Purification of remodelling enzymes
TAP (tandem affinity purification) tagged yeast strains were either purchased from Euroscarf (Germany) for CHD1 and ISW2, or in the case of RSC the strain BCY211 was kindly supplied by Brad Cairns. Six litres of yeast were grown at 30 °C to an A600 of 2–2.5 in 3×yeast extract, peptone, adenine, d-glucose and frozen by dropwise addition into liquid nitrogen. Yeast cells were lysed by mechanical disruption using a blender (Waring) kept cold by addition of solid CO2. This was then thawed and purified using standard TAP protocols77 except using higher stringency wash buffers (20 mM Na-Hepes (pH 7.4), 350 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Tween-20, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 2.6 mM aprotinin, 2 μg/ml leupeptin, 1 μM pepstatin). The purified eluate was concentrated using a Centricon YM-50 concentrator (Millipore) to 200–300 μl, dialysed against wash buffer without protease inhibitors and stored as 10 μl aliquots at −80 °C. The purity of Isw2 and Chd1 is indicated by Stockdale et al.,61 the purity of RSC complex is illustrated in Supplementary Data, Figure 8.
Native peptide ligation
H3 Δ1-27 S28C (NCBI: CAD89679) and H4 Δ1-20 V21C (NCBI: CAD89677) were generated by sited directed mutagenesis. To ensure that the N-terminal cysteine within the globular histone was available for ligation, up to 10 mg of lyophilised histone was dissolved in 1 ml 6 M guanidine chloride(GnCl) (pH 7), 10 mM DTT, and incubated at 50 °C for 30 min. This was then dialysed extensively against three changes of 4l of 10 mM sodium acetate (pH 5.2), 1 mM EDTA, allowing at least 3 h per step. The fully reduced histone was then lyophilised and stored. Modified tail peptide thioesters were purchased from CSS-Albachem (Scotland) and aliquots of peptide in use stored at 10 mM in MilliQ water at −80 °C. The ligation conditions used were similar to those used by Dawson & Kent.21,22 The 1–2 mM globular histone was dissolved in 6 M GnCl, 0.2 M phosphate (pH 7.3) together with 0.4–0.5 mM thioester peptide and 2% (v/v) thiophenol, typically to a final volume of 200–300 μl. This was vortexed, and the reaction left at room temperature for 16–24 h. The reaction was stopped by adding DTT to 100 mM and dialysing the reactants against three changes of 500 ml SAUD0 buffer (7 M urea, 20 mM sodium acetate (pH 5.2), 5 mM 2-mercaptoethanol, 1 mM EDTA pH 8.0, 0 mM NaCl) using a 12–14 kDa MWCO dialysis membrane. The dialysed ligation mixture in SAUD0 buffer was spun to remove protein precipitate, and the supernatant loaded onto a 1 ml SOURCE 15S (Pharmacia) ion exchange column running at 1.5 ml/min to separate unligated globular histone from the full length product. A stepwise elution using SAUD0 (buffer A) and SAUD2000 (buffer B) (7 M urea, 20 mM sodium acetate (pH 5.2), 5 mM 2-mercaptoethanol, 1 mM EDTA (pH 8.0), 2 M NaCl) was used to elute in as small a volume as possible. The H3 Δ1-27 S28C and H4 Δ1-20 V21C constructs elute at a conductivity of 14 milliSiemens (mS), equivalent to approximately 180 mM NaCl, whereas full length histones elute at less than 25mS, 500 mM NaCl. This is typically 9.2%, and 25% SAUD2000, but due to variations in buffer preparation, the concentration of the two buffers required to obtain this conductivity should be determined empirically. We found that histones can elute in multiple peaks even though they are identical by SDS–PAGE and mass spectrometry, presumably representing differentially folded sub-species. The globular histone was eluted over 15 ml and the full-length histone over 10 ml, before the column was washed in SAUD2000 for 6 min in-between runs to remove tightly bound proteins and then re-equilibrated in buffer A. The relevant fractions are collected, diluted with SAUD0 to a final salt concentration below 100 mM NaCl and reloaded onto the column. We found that two to three rounds of purification were required to obtain greater than 95% pure ligated protein as determined by SDS–PAGE (Gradipore, Australia). The final fractions are concentrated using a YM-10 centricon (Millipore) to approximately 200 μl and their concentration measured by absorbance at 276 nm.
Nucleosome reconstitution
Recombinant Xenopus laevis histone proteins were expressed and purified as described.78 Where necessary site-directed mutagenesis was carried out using the Stratagene Quickchange kit. Nucleosomes were assembled by mixing equimolar amounts of histone octamer and DNA in high salt and performing stepwise dialysis into low salt as described.79 DNA was generated by preparative PCR using primers obtained from Eurogentec (Belgium) fluorescently labelled where appropriate. Nucleosomes were assembled on DNA fragments based on the MMTV nucleosome A positioning sequence80 or the synthetically selected 601 sequence.81 We have adopted a nomenclature in which the lengths of DNA extensions on either side of a nucleosome are indicated as numbers on either side of a letter that defines the core positioning sequence used. So 54A18 designates a nucleosome positioned on the MMTV nuc A positioning sequence with a 54 bp extension to the upstream side and a 18 bp extension to the downstream side. The oligos used to generate the 54A18 fragment were 5′TATGTAAATGCTTATGTAAACCA and 5′TACATCTAGAAAAAGGAGC; for the 54A54 fragment 5′TATGTAAATGCTTATGTAAACCA and 5′ATCACATGTGAAAGTTAAAAAA; for the 0W0 fragment 5′CTGCAGAAGCTTGGTCCC and 5′ACAGGATGTATATATCTG; for the 54W0 fragment 5′TATGTCCATGCTCATGCC and 5′ACAGGATGTATATATCTG; for the 36W36 fragment 5′GGCGAATTCGAGCTCGGTAC and 5′AGGTCGACTCTAGAGAATCC. The PCR fragments were purified by ion exchange chromatography on a 1.8 ml SOURCE 15Q (Pharmacia) column. Radiolabelled DNA was prepared using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (Molecular Bioproducts) according to the manufacturer's specifications.
Nucleosome remodelling
Thermal remodelling reactions were performed by incubating nucleosomes in a thin-walled 200 μl PCR tube (ABgene, UK) in 50 mM NaCl, 50 mM Tris (pH 7.5) at 47 °C in a PCR machine with a heated lid for the specified amount of time. At the end of the reaction, sucrose was added to 5% (w/v) and the reactions placed on ice. The samples were separated on 0.2× Tris–Borate EDTA, 5% native polyacrylamide gels for 3.5 h at 300 V at 4 °C with pump recirculation. Gels were scanned in a Fuji Phosphoimager FLA-5100 and the bands quantified with Aida software (Fujifilm). For each time point the fraction of nucleosomes remodelled was calculated as the intensity of the destination position divided by the sum of the start and destination intensities. These data were then corrected to set the zero time point to zero percentage remodelled. The data points were plotted onto a graph and a hyperbolic curve of the form y = x/(n+x) fitted non-linearly to the data using the Solver add-in for Excel over 1000 iterative cycles. The initial rate of remodelling was calculated by differentiation of the curve and solving for time equals zero. At this point the initial rates for unmodified and modified nucleosomes within the competition assay were divided by each other to describe the initial rate of remodelling relative to the unmodified control. The average and standard deviations were calculated from at least three independent repeats. ATP-dependent remodelling reactions were performed as for thermal repositioning except using a reaction buffer containing 50 mM NaCl, 50 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM ATP. These were incubated at 30 °C with the amount of remodelling enzyme and for the time specified in the figure legends. The reactions were stopped using 500 ng of HindIII-digested bacteriophage lambda competitor DNA, adding sucrose to 5% (w/v) and placed on ice. Nucleosome binding assays were performed in 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 3 mM MgCl2 and 3% (w/v) Ficoll-400 unless otherwise stated.
ATPase assay
The ATPase assay was performed as described.31 The reaction was measured in solution in real-time on a Cary Eclipse fluorimeter (Varian, Australia). The excitation and emission wavelengths were set to 430 nm and 465 nm, respectively, with a 5 nm slit width and polarisers used to compensate for anisotropy. A calibration curve using MDCC–PBP at 5 nM and increasing amounts of inorganic phosphate was performed to determine the range across which the detection is linear. MDCC–PBP was diluted using 10 mM Pipes (pH 7) to 50 nM, this was mixed with nucleosome and enzyme in a final buffer concentration of 50 mM NaCl, 50 mM Tris (pH 7.5), 1 mM MgCl2 to a final concentration of 5 nM. Enzyme and nucleosome concentrations are as stated in the Figure legends. Measurements were performed with 1 mM ATP which had previously been treated to remove inorganic phosphate contamination.31 However, identical results were obtained using 100 μM ATP which had not been treated with the phosphate mop. The measurement of the rate of hydrolysis was performed using the Cary Eclipse software and non-linear fitting of the Michaelis–Menton equation to the data done within the Solver add-in for Excel over 1000 iterative cycles.
Octamer transfer and dimer exchange
Octamer transfer was performed under the same conditions as standard ATP-dependent remodelling. The 15 nM donor nucleosome (assembled on unlabelled 0W0 DNA) was incubated with 10 nM RSC and 1 nM radiolabelled acceptor 0W0 DNA for the specified amount of time in the presence of 1 mM ATP. The reactions were stopped by the addition of excess cold DNA and analysed by native PAGE. Thermal dimer exchange was performed as described.72 Briefly, this is performed as for nucleosome repositioning, except with a twofold molar excess of H3/H4 tetrasomes assembled onto 147 bp ‘601’ DNA.81

histone snf2 chromatin acetylation nucleosome


Adults with corrected oesophageal atresia: is oesophageal function associated with complaints and/or quality of life?

The aim of this study was to evaluate oesophageal function after correction of oesophageal atresia in adults, and to investigate the association between complaints, oesophageal function and quality of life (QoL). Twenty-five adults were included who participated in previous follow-up studies, during which complaints of dysphagia and gastro-oesophageal reflux (GOR), results of upper gastrointestinal endoscopy, oesophageal biopsies and QoL had been collected. Manometry was performed in 20 patients, 24 h pH-measurements were performed in 21 patients. pH-values (sample time 5 s) were calculated using criteria of Johnson and DeMeester. Associations were tested with ANOVA and χ2-tests. Ten patients (48%) reported complaints of dysphagia, seven (33%) of GOR. The amplitude of oesophageal contractions was low (<15 mmHg) in four patients (20%). pH-measurements showed pathological reflux in three patients (14%). Patients reporting dysphagia more often had disturbed motility (P = 0.011), and lower scores on the domains “general health perceptions” (SF-36) (P = 0.026), “standardised physical component” (SF-36) (P = 0.013), and “physical well-being” (GIQLI) (0.047). No other associations were found. This study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of oesophageal atresia. Patients reporting dysphagia, whom more often had disturbed motility, seemed to be affected by these symptoms in their QoL.
Introduction
At present, the survival rate of patients with oesophageal atresia (OA) is approximately 95% [1, 2]. With the decreased mortality, the interest in morbidity, especially the long-term results after correction of OA, has increased over the years.
Several long-term follow-up studies have shown that long lasting gastro-oesophageal reflux (GOR) is a frequent problem after correction of OA, although intestinal metaplasia, as its theoretical consequence, is rare [3–6]. In these studies, GOR has been either diagnosed by upper gastrointestinal (GI) endoscopy with biopsies, by 24 h pH-measurements, or by both, showing varying degrees of GOR. Besides pH-measurements, oesophageal manometry has been performed in several studies, showing oesophageal motility disturbances in most patients [7–10]. The true impact of this finding on individual daily life is not clear.
In our centre, we have performed several long-term follow-up studies in a relatively large group of patients after correction of OA [5, 6]. Patients underwent upper GI endoscopy with biopsies and quality of life (QoL) measurements [11].
The first aim of this study was to evaluate the presence of GOR and oesophageal motility problems in a group of our adult patients treated for OA.
The second aim was to investigate if there was an association between complaints, oesophageal function, and QoL-measurements. This association has not been investigated before. Because of the influence of complaints of dysphagia and GOR on daily life, we hypothesised that patients with complaints have a poorer QoL than patients without complaints.
Patients and methods
Twenty-five patients over 18 years of age who participated in previous follow-up studies after correction of OA [5, 6, 11], and gave informed consent to participate, were included in this study. In these previous studies, data regarding the results of upper GI endoscopy, biopsies of the distal oesophagus, and QoL had been collected. From the QoL study, we used the results of the Medical Outcome Study 36-Item Short-Form Health Survey (SF-36) and of the gastro-intestinal quality of life index (GIQLI) [12, 13]. After approval of the study protocol by the Medical Ethical Committee, all patients received a written invitation to participate in the study.
All patients who gave their informed consent, underwent manometry and pH-measurements and were asked if they experienced difficulties swallowing solid foods (dysphagia) or experienced heartburn or retrosternal pain (GOR-related complaints). Oesophageal manometry was performed using the UPS-2020 measurement stationary system (MMS, Enschede, The Netherlands) with software version 7.
The pressure was measured with the Unisensor Microtip catheter type 8304-00-9980-D with three pressure transducers on a 5 cm distance from each other. The lower oesophageal sphincter basal or resting pressure (LOSP) and relaxation after swallowing, the motility in the oesophageal body after at least six wet swallows of 5 ml water, and the upper oesophageal sphincter pressure (UOSP) and relaxation were calculated. The amplitude of oesophageal body contractions was categorized as “low” (<15 mm Hg), “moderate” (15–35 mm Hg), or “normal” (>35 mm Hg). The encountered oesophageal motility disorders were classified according to the guidelines proposed by Spechler et al. [14]. Based on the basal LOSP, LOS relaxation, peristaltic wave progression, and distal wave amplitude, oesophageal motility disorders were classified into four categories: (1) “inadequate LOS relaxation”, (2) “uncoordinated contraction”, (3) “hypercontraction”, and (4) “hypocontraction” or “ineffective oesophageal motility”.
pH-measurements were performed using the Comfortec dual channel pH probe (Sandhill Scientific), which was positioned with the pH measurement points 5 and 20 cm above the manometrically established upper border of the LOS. The position of the probe was checked by X-ray. Ambulatory pH measurement was performed during 24 h using the GORD pH-recorder (Sandhill Scientific) with a sample time of every 5 s. The pH values were calculated using the criteria of Johnson and DeMeester [15].
The data were entered into a database and analysis was performed using SPSS (Statistical Package of the Social Sciences) 10.0.1 for Windows. We tested for association between functional results and QoL by applying ANOVA and χ2-tests or Fisher’s exact tests.
Definitions
Before testing for association, the results of functional tests were dichotomized. If upper GI endoscopy showed a normal oesophagus or grade I oesophagitis (according to the modified system of Savary-Miller) [16], this was scored as “normal”. Grade II oesophagitis or worse was scored as “abnormal”. If the biopsies of the distal oesophagus showed normal oesophageal epithelium or mild reflux oesophagitis (according to Ismael-Beigi) [17], this was scored as “normal”. Moderate oesophagitis or worse was scored as “abnormal”. As all patients were diagnosed as having “ineffective oesophageal motility”, this variable could not be used. Instead, we used the amplitude of oesophageal body contractions as a measure of outcome of manometry, because decreased amplitude implies a defective peristaltic function of the oesophagus. The results were dichotomized as “normal” or “abnormal” (moderate or low amplitude). The results of pH-measurements were also dichotomized as “normal” or “abnormal” (minor or pathological reflux).
Results
Patient characteristics
Patient characteristics are described in Table 1. All patients had undergone a primary end-to-end anastomosis for Gross’ type C OA. Two patients were using proton-pump inhibitors, they stopped taking their medication two days before the start of the study.
Table 1Patient characteristics of 25 patients participating in the studyMean (range) or n (%)Age (years)28.5 (18–42)Gender  Male16 (64%) Female9 (36%)Concomitant congenital anomalies None17 (68%) Present8 (32%)  Anorectal malformations3 (12%)  Cardiac malformations4 (16%)  Vertebral malformations1 (4%)  Limb malformations2 (8%)  Other malformations2 (8%)Anti-reflux procedure in past No23 (92%) Yes2 (8%)Anastomotic stenosis in past No18 (72%) Yes7 (28%)Current educational status Primary school1 (4%) Basic high school9 (36%) Advanced high school7 (28%) University5 (20%) Unknown3 (12%)
Unfortunately, it was impossible to perform manometry and pH-measurements in four patients. In one patient it was impossible to introduce the catheter through the nose due to resistance of the patient, who decided to withdraw from the study. In three out of four patients it was impossible to introduce the catheter due to oesophageal stricture. All of these three patients had been treated for anastomotic stricture in childhood, one patient had undergone anti-reflux surgery in childhood. Two of these three patients had complaints of dysphagia at the time of the study. The stricture of these three patients was treated with dilatation. Since dilatations may influence the results of manometry and pH-measurements, no measurements were performed in these patients. Due to a technical failure, the data of the manometry of one patient could not be retrieved.
Dysphagia was reported by 10/21 patients (48%), GOR-related symptoms were reported by 7/21 patients (33%).
Manometry
The data of 20 patients could be analyzed. The upper oesophageal sphincter (UOS) responded normally to swallowing in all patients. Mean UOS pressure was 30.8 ± 15.5 mm Hg.
Oesophageal contractions were observed in all patients. One or more propulsive contractions were observed in 14/20 patients. All patients showed one or more non-transmitted contractions. Retrograde contractions were observed in 7/20 patients. Mean minimum oesophageal body amplitude pressure was 20.7 ± 13.4 mm Hg. Mean maximum oesophageal body amplitude pressure was 32.0 ± 15.5 mm Hg. The amplitude of oesophageal body contractions was low (<15 mm Hg) in 4 patients (20%), moderate (15–35 mm Hg) in 10 (50%) and normal (>35 mm Hg) in 6 (30%).
In all patients, the LOS showed complete relaxation upon swallowing. Mean LOSP was 13.1 ± 7.2 mm Hg.
According to the guidelines of Spechler et al., all patients were classified as having “ineffective oesophageal motility” [16]. The LOS pressure was normal and LOS relaxation was complete in all patients. Wave progression varied from normal to absent progression, and the distal wave amplitude was low in ≥ 30% of wet swallows (data not shown).
pH-measurements
The data of 21 patients could be analyzed. The results are described in Table 2. pH-measurements showed a normal pattern in 17 patients (81%), minor reflux in 1 (5%), and pathological reflux in 3 (14%). In both of the patients who were taking proton-pump inhibitors, pH-measurements showed pathological reflux.
Table 2 Results of 24 h pH-measurements in 21 patientsProximal mean % ± SDDistal mean % ± SDTotal time pH < 40.2 ± 0.41.5 ± 2.2Upright time pH < 40.2 ± 0.62.2 ± 3.3Supine time pH < 40.0 ± 0.30.4 ± 1.0SD standard deviation
Associations
The association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements and QoL is shown in Table 3. Patients reporting dysphagia, more often had disturbed motility (P = 0.011). These patients also had significantly lower scores on the domains “general health perceptions” (P = 0.026), and “standardised physical component” (P = 0.013) of the SF-36; and on the domain “physical well-being” (P = 0.047) of the GIQLI.
Table 3Association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements, and quality of lifeComplaintsDysphagiaGORYesNoYesNoEndoscopy Normal99612 Abnormal1212Oesophageal biopsies Normal6547 Abnormal3526Manometry Normal0*6*15 Abnormal10*4*68pH-measurements Normal107512 Abnormal0422SF-36 scales Physical functioning86.5 (19.2)94.1 (8.3)96.7 (5.2) 88.1 (16.4) Physical role-functioning77.8 (38.4)100.0 (0.0)83.3 (40.8)92.9 (20.6) Emotional role-functioning77.8 (44.1)96.7 (10.1)83.3 (40.8)90.5 (27.5) Bodily pain78.2 (26.3)92.5 (13.9)85.5 (24.5)86.3 (20.5) General health63.6 (23.6)**84.6 (15.1)**86.7 (14.8)70.2 (22.7) Vitality50.0 (15.4)52.3 (12.9)58.3 (13.3)48.2 (13.2) Social functioning88.9 (22.0)93.2 (18.8)97.9 (5.1)88.4 (23.2) Mental health82.7 (15.781.5 (16.7)87.3 (12.5)79.7 (17.0)SF-36 component summary scales PCS49.5 (7.4)***56.4 (3.4)***55.6 (4.9)52.3 (6.9) MCS49.0 (10.9)49.5 (7.4)50.3 (7.9)48.8 (9.5)GIQLI scales Physical well-being21.2 (3.1)****24.5 (3.7)****23.3 (3.8)22.9 (3.9) GI symptoms64.1 (8.5)68.6 (7.8)68.0 (6.5)66.0 (9.0) Social well-being12.0 (1.7)12.2 (0.4)12.0 (0.6)12.1 (1.3) Emotional well-being15.1 (1.5)15.9 (0.5)15.5 (1.0)15.6 (1.2)Total score112.4 (13.3)121.3 (9.6)118.8 (10.3)116.6 (12.9)Data are shown as n, or mean (standard deviation)GOR gastro-oesophageal reflux, SF-36 Medical Outcome Study 36-Item Short Form Health Survey, PCS physical component summary, MCS mental component summary, GIQLI gastro-intestinal quality of life index*P = 0.011, **P = 0.026, ***P = 0.013, ****P = 0.047
No association was found between complaints of GOR and quality of life; nor between the results of endoscopy and the results of pH-measurement and/or manometry; nor between the results of oesophageal biopsies and the results of pH-measurement and/or manometry (data not shown). Problems with the initial surgical repair in childhood (i.e. anastomotic leak or stricture needing dilatation) did not influence the findings in the current study.
Discussion
The results of the present study confirm that oesophageal motility disturbances are frequently present after correction of OA. Low or moderate amplitude of oesophageal body contractions were found in 14/20 patients (70%), all patients showed one or more non-transmitted contractions, and retrograde contractions were observed in 7/20 patients (35%). All patients met the manometric features of “ineffective oesophageal motility”, as described by Spechler et al. [14]. The manometric findings in the present study are comparable to those described in other studies [7–10].
Based on pH-measurements, the prevalence of GOR in the current patient group is lower than expected. pH-measurements showed minor or pathological reflux in 4/20 patients (20%). None of these patients had undergone anti-reflux surgery in the past. In other studies, the prevalence of GOR based on pH-measurements varies from 17 to 54% [7–9]. Unfortunately, the criteria used for diagnosing GOR and the age of the patient groups studied also vary between studies.
The question is, what the influence is of the disturbed oesophageal motility and GOR found in previous studies on the daily life of adults with corrected OA. This is the first study in which complaints and QoL after correction of OA have been combined with long-term studies of oesophageal function: endoscopy, oesophageal biopsies, manometry and pH-measurements. Patients reporting dysphagia more often had disturbed motility, and showed significantly lower scores on the domains “general health perceptions” and “standardised physical component” of the SF-36, and on the domain “physical well-being” of the GIQLI. However, GOR-related complaints were not associated with disturbed oesophageal function, and did not influence QoL. It is important to consider that this group of patients has grown up with these symptoms, and may probably have gotten used to it. The fact that complaints of dysphagia affect the QoL and GOR-related complaints do not affect QoL may be explained by the influence of these complaints on daily activities such as eating.
Motility problems after correction of OA were first reported by Haight [18] in 1957. The main cause of the abnormal oesophageal motility after correction of OA is not clear. Some studies propose a congenital nervous abnormality as the cause of motility disturbances. In the foetal rat model for OA, abnormalities were found in the course and branching pattern of the vagal nerves [19]. However, an acquired cause is also suggested, i.e. surgical damage to vagal fibres that innervate the oesophagus [20]. Abnormal oesophageal motility can cause symptoms of dysphagia. It can also worsen the effects of GOR, since malfunction of the peristaltic pump will result in a delayed clearance with a longer period of stasis of refluxed material in the oesophagus.
In conclusion, this study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of OA. Only patients reporting dysphagia, who more often had disturbed motility, appeared to be affected by these symptoms in their QoL.

oesophageal atresia
quality of life
manometry
ph-measurements
long-term follow-up


Deficit of social cognition in subjects with surgically treated frontal lobe lesions and in subjects affected by schizophrenia

The ability of humans to predict and explain other people’s behaviour by attributing independent mental states such as desires and beliefs to them, is considered to be due to our ability to construct a “Theory of Mind”. Recently, several neuroimaging studies have implicated the medial frontal lobes as playing a critical role in a dedicated “mentalizing” or “Theory of Mind” network in the human brain. In this study we compare the performance of patients with right and left medial prefrontal lobe lesions in theory of mind and in social cognition tasks, with the performance of people with schizophrenia.
Introduction
Social cognition refers to the ability to perceive, interpret and provide an adequate response to affective and other interpersonal cues [1]. Pioneering studies have found that schizophrenic subjects show social cognitive impairments, in particular in modifying their behaviour when interacting with other people and in recognizing social information cues [2]. People with schizophrenia also show a great deal of social naivety in interpersonal situations [3].
The ability to engage in competent social relations and to understand social information depends on the adequate functioning of a mental mechanism termed Theory of Mind (ToM), that allows people to understand and interpret their own and other people’s mental states and hence to predict and explain their behaviour [4, 5]. Evidence from neuroimaging and neuropsychological studies has led researchers to conclude that ToM is subserved by dedicated brain systems, including the amygdala, the temporo-parietal junction, the orbital frontal cortex and, in particular, the medial frontal lobes [6, 7]. The results of these studies have been used to argue that the medial frontal lobes play a critical role in a dedicated mentalizing system [7, 8].
Neuropsychological literature specifically relevant to the medial frontal cortex is scarce. Actually, only three studies have included patients with relatively focal medial frontal lobe damage and have sought to investigate correlations between specific frontal brain areas and performance in ToM tasks [9–11]. However, there are important limitations to these investigations. Stuss et al. [10] reported that patients with right frontal and bilateral frontal lobe damage were impaired in their ability to infer visual experience in others. The authors conclude that acquired brain damage to the medial frontal lobes does impact on ToM ability, but aetiology of brain damage for each patient is not reported, the text implies that most of the bifrontal group had suffered head traumas. It is often very difficult to assess the extent of brain damage reliably after head trauma as widespread damage can often occur through axonal shearing and other effects (e.g. [12]). The second neuropsychological study [9] reports that patients with left and right-sided lesions were equally impaired, while lesion size was unrelated to performance and no effect of lesion location was found when comparing patients with focal dorsolateral, medial or orbital frontal lesions.
Also, a recent group study of empathy in patients [11] included an assessment of performance on the Faux Pas Test, a probe of ToM ability. Patients with frontal lobe lesions were impaired in this task. Specifically, patients with ventromedial frontal lobe damage made significantly more errors than patients with posterior lesions or healthy controls in the Faux Pas Test.
However, a detailed analysis of lesion sites associated with ToM deficits in these previous studies revealed a particularly important role for the right ventromedial prefrontal frontal lobe [11, 13]. Damage in these areas induces behavioural changes affecting personality (indifference), impaired social judgement, reduced affect and goal-directed behaviour, self-monitoring deficits [14–16].
More recent studies have provided evidence that is in disagreement with a single area hypothesis involved in ToM processing. In fact, the neural network involving the right and left temporo-parietal-junction (TPJ R and L); the posterior cingulated (PC), and medial prefrontal cortex (MPFC) seems to be crucial in processing the complex reasoning involved in mentalizing [17, 18]. Bird et al. [19] studied a patient affected by extensive damage of the medial frontal lobes reporting, interestingly, a dysexecutive syndrome with confabulation with preserved performance on some ToM tasks.
Saxe and Wexler [20] in an fMRI study in normal subjects, described an equally selective profile of activation of the above-mentioned areas in a multicomponent pattern of activation fMRI methodology. Frontal lobe damage in patients has long been linked to impairments in social behaviour [21]; in fact, they have been described as presenting diminished social awareness and a lack of concern for social rules [15, 22].
An interesting approach to social competence deficits in schizophrenic people may be represented by the Machiavellian Intelligence Hypothesis (MIH, [23, 24]).
According to this hypothesis, in the development of intelligence, social, rather than technical, efficiency represents the main selective pressure of human evolution [25]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the ventromedial prefrontal cortex: selective damage to these areas causes a relevant impairment of interpersonal relationships and in regulating behaviour according to social rules [27–30].
Patients with lesions to the orbitofrontal cortex also have disinhibited/socially inappropriate behaviour. Grafman interpreted the patient’s impairment in terms of an inability to access “social schema knowledge” stored in the frontal lobes [31]. Social schema knowledge is thought to inhibit aberrant behaviour. Patients with orbitofrontal cortex lesions who cannot access social schema knowledge fail to inhibit aberrant behaviour, such as physical threats and aggression.
The prediction of a similar cognitive profile in terms of ToM abilities and social competence between frontal lesion subjects and schizophrenic people has been investigated and confirmed [6, 7]. An extensive and careful review recently published [32], reported a general agreement about the nature and extension of ToM dysfunctions in people affected by schizophrenia. These dysfunctions are symptoms related [33], disease specific and state independent.
The neural architecture of the social cognitive dysfunction of schizophrenia is of paradigmatic importance for the understanding of social cognitive dysfunction and, more importantly, for the understanding of the consequences at the behavioural level [34]. A previous seminal study provided evidence that structural orbitofrontal cortex abnormalities are related to social dysfunction in schizophrenic people [35]. Moreover this prefrontal area has been unequivocally involved in the social cognitive deficits associated with this disorder [36].
However, empirically controlled investigations in which the cognitive profile of brain damaged patients was compared with schizophrenic subjects with an appropriate set of ToM and social intelligence tasks are lacking, thus leaving several crucial questions largely unresolved.
In the current study, we examined the performance of stabilized schizophrenic outpatients, inpatients with focal damage of left and right ventromedial prefrontal lobes and healthy controls, in ToM abilities, in social competence and tactical strategy (Machiavellian Intelligence), to clarify whether schizophrenic patients demonstrate impairment similar to ventromedial prefrontal lesion patients and whether their performance in these tasks can be differentiated from their performance in tasks sensitive to neuropsychological dysfunction, including “executive” functions. Our prediction is that an overlapping dysfunctional cognitive profile should emerge between brain damaged and schizophrenic subjects, when compared to healthy subjects. We also addressed several methodological issues raised by earlier studies using social cognition tasks created for adults and not for children.
Methods
Participants
The subjects for this study included 18 adult neurosurgical patients with unilateral frontal lobe lesions who had undergone surgery at the Department of Neurosurgery of “S. Salvatore” Hospital, L’Aquila, Italy. The patients were consecutively enrolled in the study from January 2003 to September 2005.
Only patients with exclusive frontal lesions were identified and brain damage was confirmed through neuroimaging, with pre- and postoperative CT scanning and MRIs.
Nine subjects with left side medial prefrontal cortex (LMPFC) lesions and nine subjects with right side medial prefrontal cortex (RMPFC) lesions were studied and underwent neuropsychological examinations (Fig. 1A, B).
Fig. 1(A) MRI T1 weight (axial view) with gadolinium showing low density abnormalities (meningioma excised, patients L.F.) orbitofrontal cortex on the left side. (B) CT scan image showing low density abnormalities (meningioma excised, patients L.G.) orbitofrontal cortex on right sides
Thirteen subjects had intra- or extra-axial tumours (72%); 3 had spontaneous haemorrhage (17%) and 2 (11%) had intracerebral haemorrhage from ruptured aneurysms.
In the RMPFC group seven patients had a tumour removed (four had a meningioma excised, 2 had a high grade glioma excised and 1 had an oligodendroglioma excised), one had a right anterior communicating artery (ACoA) aneurysm clipped, following rupture and one had spontaneous intracerebral haemorrhage with no evidence of arterial malformations. Of the LMPFC patients, six had a tumour removed (five had a meningioma excised and one had a high grade astrocytoma excised), two had spontaneous intracerebral haemorrhage with no evidence of arterial malformations and one had a left ACoA aneurysm clipped following rupture.
The location of the experimental group’s lesions were defined anatomically as medial (Broadman area 9 and 46) and orbital (Broadman areas 10, 11, 12 and 25) and further classified according to the prefrontal sectors of functional significance into which the lesions encroached (Tables 1, 2).
Table 1Socio-demographic details of subjects with ventromedial frontal lobe lesionsSubjectsSiteAgeEducationAetiologyM.DRight frontal lesion5613MeningiomaF.S.Right frontal lesion508MeningiomaL.GRight frontal lesion448MeningiomaP.G.Right frontal lesion635OligodendrogliomaT.RRight frontal lesion625Glioma grade IIIE.GRight frontal lesion698Spontaneous intracerebral haemorrhageS.MRight frontal lesion6413Glioma grade IVA.URight frontal lesion3518Intra-cerebral haemorrhage from aneurismal sac ruptureM.URight frontal lesion4418MeningiomaL.FLeft frontal lesion418MeningiomaL.PLeft frontal lesion305MeningiomaM.NLeft frontal lesion6813Spontaneous intra-cerebral haemorrhageM.M.Left frontal lesion5513Spontaneous intra-cerebral haemorrhageR.MLeft frontal lesion548MeningiomaD.SLeft frontal lesion628MeningiomaR.TLeft frontal lesion7313Astrocytoma grade IIIA.CLeft frontal lesion598MeningiomaR.GLeft frontal lesion388Intra-cerebral haemorrhage from aneurismal sac ruptureTable 2Classification of the RMPFC and LMPFC experimental group according to the prefrontal sectors of functional significance into which the lesions encroachedPatientSexAge (years)AetiologyLesion locationFronto-orbitalMedialRF1F56Meningioma+RF2F50Meningioma+RF3M44Meningioma+RF4F63Oligodendroglioma+RF5M62Glioma grade III+RF6M69SIH+RF7F64Glioma grade IV+RF8F35IhAsc+RF9M44Meningioma+LF1M41Meningioma+LF2M30Meningioma+LF3F68SIH+LF4M55SIH+LF5M54Meningioma+LF6M62Meningioma+LF7M73Astrocytoma grade III+LF8F59Meningioma+LF9M38IhAsc+SIH = spontaneous intracerebral haemorrhage; IhAsc = intracerebral haemorrhage from aneurismal sac rupture
Subjects were assessed with neuropsychological test batteries 20–40 days after surgery.
A control group of schizophrenic subjects and a control group of psychiatrically and neurologically healthy subjects were studied as well.
Twenty male patients, all native Italian speakers, whose symptoms satisfied the DSM-IV criteria for schizophrenia [37], participated in the study. They were all patients of the Department of Psychiatry of the University of L’Aquila and diagnosis was re-confirmed on admission to (and 6 months after discharge from) the Day-Hospital (DH) using a non-structured interview conducted by two psychiatrists (M.C., R.R) referring to DSM-IV criteria.
All the subjects, whose assessment took place when clinically stable within a month of admission to the DH and establishment/confirmation of diagnosis, were treated with maintenance antipsychotic drugs. The mean daily dose was 310.3 (SD 143.67) mg/equivalents of Chlorpromazine [38]; this dose-equivalence with a typical antipsychotic is necessary in order to compare the different antipsychotic drugs as their strength may be different when administered to patients.
Twenty neurologically and psychiatrically healthy control subjects (matched for age and education) were included. Exclusion criteria were: history of neurological disease including epilepsy, head trauma or mental retardation. All subjects provided informed consent to participate in the study.
Materials and procedure
Clinical assessment
Clinical assessment in the sample with MPFC lesions was performed by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 translated into Italian by [39] and through the neuropsychiatric Inventory (NPI), a semistructured clinicians interview using the protocol described by [40].
For the schizophrenic sample, frequency and severity of current symptomatology was registered by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 (Modified 24-item version, translated into Italian by [39] and Clinical Global Impression scale (CGI) [40].
The prevalent symptomatology was also investigated using the Scale for the assessment of Positive Symptoms (SAPS; [41]) and Scale for the assessment of Negative Symptoms (SANS; [42]).
The SAPS consists of 34 items and is divided into four subscales: hallucinations, delusions, bizarre behaviour and formal thought disorder. The SANS consists of 25 items and is designed to measure five domains: affective flattening or blunting, alogia, apathy, asociality and impaired attention.
We also evaluated social function with AD-Disability Assessment [40]. Socio-demographic and clinical data are reported in Table 3.
Table 3Socio demographic, clinical details in the total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Sex (M:F)7:135:42:715:6Age (years)42.7 (1.8)49.6 (10.3)46.4 (18.6)38.9 (2.8)Education (years)8.8 (0.8)9.2 (2.6)12.2 (3.97)13.4 (1.2)Day since surgery–91.4 (11.6)88.8 (13.7)–IQ level88.8 (5.7)93.1 (2.4)98.7 (3.6)–NPI total score6 (0)10 (0)9.8 (5.3)–Duration of illness (years)12.30 (4.13)–––BPRS total score44 (11.1)52.5 (4.9)55.5 (17.5)–C.G.I.4.5 (0.6)–––SANS50.42 (20.9)–––SAPS42.57 (19.1)–––Social function (AD)2.5 (1.1)–––The values are means; standard deviation are in parenthesis
Neuropsychological assessment
All the schizophrenic subjects, with the exception of two positive schizophrenics, were also administered three neuropsychological tests for assessment of executive functions: the WCST, the Tower of London Test and a Phonemic Verbal Fluency Test (Table 4).
Table 4Neuropsychological detailed of total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Verbal fluency33.1 (10.4)21.2 (8.39)11.3 (4.19)38.7 (7.5)Tower of London29.8 (4.4)26.3 (6.5)19.2 (4.3)34.6 (1.9)WCST no. of category3.46 (2.41)2.66 (1.7)1.87 (2.1)5.7 (0.3)WCST % of perseverative errors27.66 (15.12)42.9 (22.6)50.7 (21.3)2.9 (2.6)Verbal memory19.1 (8.7)28.6 (8.01)8.3 (2.25)29.6 (3.01)The values are means; standard deviation are in parenthesis
The WCST (128 cards) was administered with standard instructions, as described by Spreen and Strauss [43], whereas scoring followed Heaton’s [44] rules. Scoring and administration instructions for the Tower of London Test were those described by Krikorian et al. [45]. Scoring and administration instructions of the Phonemic Verbal Fluency Test were those described by Novelli et al. [46].
Visuo-spatial intellectual level was assessed by means of Raven’s Progressive Matrices [47].
Theory of mind tasks
Four stories were read to the participants individually to assess ToM competence. The stories were structured to assess the ability to understand first and second order false beliefs in adult subjects [9]. First order false beliefs require a subject to make an inference about the state of the world. To assess first order ToM two stories were used: The washing machine task [9] and The Cigarette Task [48].
Second order stories measure the capacity to understand other people’s false beliefs. To assess second order ToM two stories were used; The Burglar [49] and The Wallpaper Story [9]. These stories were presented to the subjects in a series of cartoons in which the various actions of the characters are depicted in sequences.
All the subjects were asked a ToM question and three control questions:
False belief test question. This was designed to elicit a response that demonstrated the ability to make inferences about another individual’s mental state, namely, that a character in the story holds a false belief.Fact question. This was posed to determine whether subjects understood the actual sequence of relevant events that had occurred in the story which is in contrast with the sequence as understood by one of the characters in the story and that leads to his coming to a false conclusion.Memory question. This was used to assess whether memory for story details was approximately intact. The stories were the same for all the subjects examined. Each subject obtained a score ranging from 0 to 1 in the case of a correct answer to a False-belief question, to the fact question and to the memory question, where 0 = incorrect answer; 1 = correct answer. If the subject gave a correct answer to both the first order stories, s/he had a global score for first order ToM equal to 1 (non-casual performance). For second order false belief stories we followed the same methods.
Social cognition tasks
The following tasks addressed two aspects of social cognition.
The ability to process the appropriateness of behaviour in different social contexts [21]; and the ability to use tactical strategy (Machiavellian Intelligence).
Social situations task
This task investigates the capacity to judge the appropriateness of behaviour that may induce anger in observers.
Procedure. Nine short stories describing social situations incorporating behaviour were read by the patient. At various points in each story, the patient was asked to comment on how appropriate the behaviour was, giving a score from A to D. “A” scores meant that he judged the situation as normative. “B” to “D” scores meant that he judged the situation as a norm violation and indexed the extent of the violation (“B” scores being mild and “D” being serious). Blair and Cipollotti [21] report that previous piloting on a large, independent sample of healthy controls had resulted in the identification of a set of consistently identified normative situations and violations.
Two scores were obtained for this task: one referring to the number of normative situations and the other to the number of violations correctly identified. The third refers to the extent to which the patient judged the violations to be socially inappropriate.
For each situation, the participant obtained a score between 0 and 3, matching their response of “A” to “D” (i.e. “A” = 0, “D” = 3).
Mach IV Scale
We used the Mach IV Scale to assess the “Machiavellian Intelligence” of participants [50]. This is a self-report Likert scale, with scores ranging from 1 to 7 (where 1 = I totally disagree; 4 = no opinion; 7 = I totally agree), composed of 20 items, each consisting of a statement.
The Mach IV scale is a method for assessing awareness and social functioning in a social context characterised by interpersonal deception (“Machiavellianism”).
From the 20 statements of the Mach IV Scale we extracted two groups of items: (1) 5 items describing duplicity tactics, e.g.: “It is wise to flatter important people” (“tactics+”) and (2) 4 items describing a disagreements with tactics duplicity, e.g.: “When you ask someone to do something for you, it is best to give the real reason” (“tactics−”).
The items were labelled for coding as follows:
Positive tactics (tactics+): subjects must agree with statements reporting the ability to manipulate other people’s intentions and actions, according to Niccolò Machiavelli’s beliefs.
Negative tactics (tactics−): subjects must agree with statements reporting judgements of correct and honest behaviour; for this reason they cannot use “intentional deception” mechanisms involved in the Machiavellian Intelligence Hypothesis. These features depend on an accurate interpretation of even the most particular intention of respondents [32, 51].
Human nature components referring to “people’s knowledge”, in particular the degree of cynicism concerning other people’s intentions and decisions are strictly related to the ability to interpret other people’s mental states.
Statistical analysis
One-way ANOVA was used to compare demographic, clinical information and neuropsychological Assessment and social cognition tasks. The Kruskaal ± Wallis test was used to analyse the level of significance of patients’ scores on ToM tasks.
Results
No differences among groups emerged for age [F(3,40) = 0.271, P = 0.751], years of education [F(3,38) = 0.387, P = 0.51] and estimated IQ [F(3,38) = 0.44, P = 0.64] (Table 2).
Clinical assessment
Neither were differences found between the four subgroups in age [F(3,38) = 0.271, P = 0.751, sex ratio, standard of education [F(3,38) = 0.387, P = 0.51], IQ [F(3,38) = 0.387, P = 0.51], in BPRS total scores [F(3,38) = 0.345, P = 0.72] and in NPI total score [F(3,38) = 0.748, P = 0.508].
Executive function
The three groups differed significantly in the planning Tower of London task [F(3,40) = 22.568, P = 0.000]; Verbal Fluency [F(3,40) = 40.023, P = 0.000], and No. of categories achieved in the WCST [F(3,40) = 9.578, P = 0.000] and perseverative errors [F(3,40) = 10.694, P = 0.000]. The performance data for the four groups on the tests of executive functioning are shown in Table 3.
The LSD method was used for post hoc comparisons. This revealed that in Verbal Fluency both frontal groups (LMPFC and RMPFC) performed significantly worse than the schizophrenic group and control subjects. Bonferroni tests on the Tower of London task showed impaired performance for the LMPFC group only when compared to both the schizophrenic and healthy subjects control groups. The LMPFC group performed significantly worse than the RMPFC subjects.
First-order false belief tasks
False-belief test question: Groups differed significantly on the non-parametric Kruskall–Wallis test: χ2(3) = 14.664, df = 3, P < 0.002. Post hoc (Bonferroni methods) comparisons showed that the RMPFC group’s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences = −0.665; P < 0.005) but not from schizophrenic subgroups, and that the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group.
Fact questions: The percentage of correct scores revealed no significant overall differences between groups.
Memory questions: The percentage of correct scores showed no significant group differences (Fig. 2).
Fig. 2Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects, Left Medial Prefrontal Cortex—LMPFC Lesion Subjects and healthy controls) on First-order False Belief tasks
Second order theory of mind
False-belief test question: Groups differed significantly: Kruskall–Wallis χ2(3) = 11.72, df = 3, P = 0.008). Post hoc (Bonferroni methods) analyses revealed the most impaired performance for the RF and schizophrenia groups when compared to LMPFC (LMPFC vs. RMPFC mean differences = −0.598; P < 0.007; LMPFC vs. schizophrenics mean differences = −0.623; P < 0.000). However the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group.
Fact questions: The percentage of correct scores revealed no significant overall difference between the groups.
Memory questions: The percentage of correct scores showed no significant group differences (Fig. 3).
Fig. 3Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects, Left Medial Prefrontal Cortex—LMPFC Lesion Subjects and healthy controls) on second-order False Belief tasks
Social situation task
The ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences in the ability to identify normative situations [F(3,58) = 3.179, P = 6.073].
Post hoc (Bonferroni methods) comparisons showed that the RMPFC group’s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences = −1.904; P < 0.001; LMPFC vs. schizophrenics mean differences = 1.623; P < 0.002) and healthy controls but not from schizophrenics. There were no significant differences between the four groups in the total score of the norm violations (Fig. 4).
Fig. 4Performances on social situation task in all groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects and Left Medial Prefrontal Cortex—LMPFC Lesion Subjects). The figure reports the scores in correct identification of normative situation and the mean scores of norm violations
Mach IV scale
The ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences on the following Mach IV items (tactics+) item “Trusting someone means getting into trouble” [F(3,58) = 3.179, P = 0.035]; (tactics+) “It’s hard to be successful without taking short-cuts” [F(3,58) = 3.959, P = 0.000]; (tactics−) “There is no need to deceive anyone” [F(3,58) = 7.759, P = 0.021]; (tactics−) “It is possible to be good in all situations” [F(3,58) = 3.406, P = 0.027]. Post hoc multiple comparison (Bonferroni methods) showed that RMPFC score lower for items indicating agreement in strategic thinking (tactics+); than LMPFC, schizophrenics and controls showed higher scores for items indicating disagreement with strategic thinking (Tactics). Results are displayed in Fig. 5.
Fig. 5Performance on items of Mach IV Scale in the four groups for strategic thinking
Correlation analyses
No significant correlations were found between ToM first order and ToM second order questions and executive functions (verbal fluency, WCST number of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right frontal lesions.
No significant correlations were found between the Mach IV scales (good tactical strategy and negative tactical strategy) and executive functions (verbal fluency, WCST no. of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right MPFC.
There was a significant correlation between first order ToM scores and duration of illness (r = −0.375; P < 0.029); between first order ToM scores and SANS total scores (r = −0.562; P < 0.03); between first order and second order ToM scores and social functioning total scores (AD) (first order = −0.489; P < 0.036; second order r = −0.543; P < 0.029).
The significant correlation was also found in the schizophrenic sample between ToM performances and verbal fluency (r = −0.527; P < 0.000).
No significant correlations were found between the Mach IV scale and social cognition task and psychopathological and clinical variable (SANS, SAPS and CGI).
Discussion
One of the distinctive attributes of human social cognition is our propensity to build models of other people’s minds: to make inferences about the mental states of others. Several neuroimaging studies have attempted to elucidate the neural substrates that support this distinctively human ability that is impaired in people with schizophrenia.
The main aim of the present paper is to establish whether patients affected by schizophrenia show an impairment in several social cognitive tasks as demonstrated in other researches [5, 52–54] and if this cognitive profile is comparable to patients with a unilateral brain lesion involving orbito-ventromedial areas of the frontal lobes.
Our results are in line with other studies: in people with schizophrenia there was an impairment of social cognitive abilities and this deficit appears to be related to negative symptomatology [55] and to be a key determinant of functional outcome, including social outcome [56]. It has been suggested that theory of mind deficit make unable schizophrenic subjects to interact effectively with their social environment, but that a lack of certain aspects of social cognition will lead to social misperceptions.
In addition to these clinical and outcome goals, there is increasing interest in identifying the neural substrates that underlie social cognitive deficits in schizophrenia. For all of these reasons we compared the performances on social cognition tasks of schizophrenic subjects with the performances of MPFC subjects.
Among the several studies which investigated the effects of frontal lobe lesions (dorsolateral and ventromedial/orbital) on performance in ToM tasks [9–11, 57–59], some of these including patients with bilateral frontal lobe damage, are limited because of a lack of detailed anatomical specification of lesion location [10, 11, 57]. Moreover, most of patients with bilateral lesions had suffered head trauma, an aetiology associated with rather diffuse brain damage that is particularly likely to impinge on orbitofrontal brain areas.
Thus, the present study strictly tests the hypothesis that the unilateral (right or left) medial frontal cortex is implicated in the neural network sub serving ToM [8] which is based on well established evidences suggesting the implication of the ventromedial frontal lobe areas in playing a critical role in a dedicated “mentalizing” or ToM network in human brains ([7, 28, 60] for a review).
We found out that subjects with RMPFC lesion are impaired in ToM tasks of “false beliefs”, showing thus a very similar cognitive dysfunctional profile to people affected by schizophrenia in all Theory of Mind tasks and in all social cognition tasks. A normal performance on control questions indicates an unimpaired comprehension of stories and suggest that the task was sensitive in detecting TOM impairments.
In addition, schizophrenics and subjects with RMPFC lesion also showed impairment in the social cognition tasks, in fact they both failed to discriminate in judging inappropriate behaviour likely to induce anger in observers. This was unlike patients with LMPFC who showed no impairment on any of these tasks.
This is clear evidence that the medial frontal cortex plays a critical role in a dedicated “mentalizing” brain network that underpins ToM ability [7, 8].
Our findings are in agreement with previous “lesional” studies, showing the association between right medial area damage and more severe ToM deficits [11, 13]. Siegal et al. [13] reported that ToM impairments seem to be associated with right hemisphere damage.
In the present study we report a dissociation in RMPFC damage patients who displayed a defective ToM performance in contrast to LMPFC patients. LMPCF subjects show lower performances than RMPCF subjects in other cognitive competences but have normal performance in ToM competences and our results confirm the results obtained by Siegal and Surian [61].
In addition, when a more sophisticated social ability is required in order to perform second order false belief tasks correctly, also LMPFC damaged subjects fail to perform at a normal level and show a statistically significant impairment, even though to a lesser degree, when compared to subjects with RMPFC and schizophrenic people. A possible explanation is that LMPFC is involved in more sophisticated mentalizing tasks and that an intact right hemisphere structure is nevertheless required [58]. Normal subjects and neurosurgical subjects with unilateral LMPFC lesions perform fairly well on tasks related to tactical strategy, showing correspondingly low scores on the ingenuity aspect of thinking.
The present study also provides further data on the neural prefrontal areas involved in social cognition tasks and in strategic thinking [21]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the frontal cortex [27, 28]; the ability to recognise and manipulate hierarchical states to achieve some advantage would be localised in the amygdala and right hemisphere [62].
Schizophrenic people and subjects with RMPFC lesion showed impaired performance on tactical strategy associated with relatively “high levels” of social ingenuity and have an impaired ability to access “social schema knowledge” which is stored in the frontal lobes [31]. Such patients cannot access social schema knowledge and fail to inhibit aberrant behaviour, such as physical threats and aggression [21, 63]. Interestingly a completely reversed pattern characterizes the performance of LMPFC lesion subjects and healthy controls, in fact we found out a complete disassociation of the neural prefrontal areas located in the medial part of the hemisphere in sub serving human ability to think strategically, indicating that the cortical organization related to tactical aspects of Machiavellian Intelligence is lateralized to the right hemisphere [64]. Our results are at slight variance with the pioneering study of Rowe et al. [9] who found a significantly impaired performance of both the RMPFC and LMPFC subjects in first and second order ToM tasks. In this study cortical lesions were non-exclusive medial hemisphere but includes subjects with dorsolateral prefrontal cortex (DLPC) damage.
Studies of normal subjects have used a variety of imaging techniques, designs, and test materials, but especially PET and fMRI to define brain regions specifically activated during a ToM task [8, 65]. Such studies have consistently shown activation of the medial prefrontal gyrus (MPFG) and the Temporo Parietal Junction.
Frith and Frith [7], reporting data on studies carried out in adults, have revealed an MPFC system of three components that are consistently activated during both implicit and explicit mentalizing tasks. This brain region is probably the basis of the decoupling mechanism that distinguishes mental state representations from physical state representations. We can speculate, according to the Edelman model [66, 67], that a hierarchical organization of mental operation, when disrupted at a specified level, impairs the integrity of final output via an interruption of the chain of events required to perform a task. This study is limited by the small number of patients with unilateral frontal lobe lesions due to the rarity of such lesion.
Despite this limitation, this study provides further evidence that social competence is compromised in RMPFC subjects very closely to schizophrenics and these data seem to elucidate the possible neuroanatomic structure alteration present in schizophrenia. However, we are confident that there is a wide range of behavioural manifestations of frontal lobe dysfunction, and ToM impairments clearly cannot account for all of these, nor is it likely to be responsible for all reported difficulties in social cognition. In contrast, ToM tests are designed with the aim of isolating those aspects of social cognition associated with two-way reciprocal interactions that rely crucially on ToM ability and false belief tasks have facilitated the demonstration of a mentalizing impairment in subjects with lesions of the prefrontal cortex, which is independent of non-mental state inference.
In conclusion, our findings provide evidence that lesions to the right MPCF determine an incapacity to understand ToM “false belief” stories and to use tactical strategy and understanding of social schema. Other authors have reported the same results: subjects with right hemisphere damage, but not subjects with left hemisphere damage, had difficulties in performing simple theory of mind tasks [13, 68].
The difficulties shared by subjects with right hemisphere damage and young children on ToM tasks may have a similar origin [61]. These may both derive from a pragmatic deficit that prevents subjects from interpreting the implicit questions correctly, rather than from a conceptual deficit concerning the ability to represent mental states.
The response pattern of subjects with ventromedial prefrontal damage on ToM tasks adds new evidence to the growing literature on the effects of the right hemisphere on various pragmatic aspects of language production and comprehension [13].
The good performance of subjects with RMPCF lesions in executive function and verbal memory tasks suggests that their difficulties are due to a reduced sensitivity to the constraints that guide the interpretation and production of contextually appropriate utterances [61].
It may be concluded that the ToM disorder in RMPCF subjects does not stem from executive function or memory deficits and that deficits in ToM and executive functioning in subjects with frontal lobe lesions are not causally related, even though our findings are in disagreement with Channon and Crawford [69] who found a relationship between executive functioning and ToM ability in adults with damage to the frontal lobes [70].
We support two positions: first, that a specialized, discrete ToM module, or set of modules, is located in the frontal lobes, but is functionally independent and second that these deficits can co-occur, on the basis of the proximity of the respective underlying neural areas.

social cognition
frontal lobe lesions
schizophrenia
theory of mind