 Research
 Open Access
 Published:
Topological structure analysis of chromatin interaction networks
BMC Bioinformatics volume 20, Article number: 618 (2019)
Abstract
Background
Current HiC technologies for chromosome conformation capture allow to understand a broad spectrum of functional interactions between genome elements. Although significant progress has been made into analysis of HiC data to identify biologically significant features, many questions still remain open, in particular regarding potential biological significance of various topological features that are characteristic for chromatin interaction networks.
Results
It has been previously observed that promoter capture HiC (PCHiC) interaction networks tend to separate easily into welldefined connected components that can be related to certain biological functionality, however, such evidence was based on manual analysis and was limited. Here we present a novel method for analysis of chromatin interaction networks aimed towards identifying characteristic topological features of interaction graphs and confirming their potential significance in chromatin architecture. Our method automatically identifies all connected components with an assigned significance score above a given threshold. These components can be subjected afterwards to different assessment methods for their biological role and/or significance. The method was applied to the largest PCHiC data set available to date that contains interactions for 17 haematopoietic cell types. The results demonstrate strong evidence of wellpronounced component structure of chromatin interaction networks and provide some characterisation of this component structure. We also performed an indicative assessment of potential biological significance of identified network components with the results confirming that the network components can be related to specific biological functionality.
Conclusions
The obtained results show that the topological structure of chromatin interaction networks can be well described in terms of isolated connected components of the network and that formation of these components can be often explained by biological features of functionally related gene modules. The presented method allows automatic identification of all such components and evaluation of their significance in PCHiC dataset for 17 haematopoietic cell types. The method can be adapted for exploration of other chromatin interaction data sets that include information about sufficiently large number of different cell types, and, in principle, also for analysis of other kinds of cell typespecific networks.
Background
The basic functionality of a genome is regulated on many levels, especially regarding how and when individual genes are transcribed and subsequently translated into proteins. The timely, specific and accurate regulation of these processes is the key to the proper functioning of an organism. Most of an organism’s regulatory processes originate in its own genome, two primary categories of these processes can be delineated based on whether they are interactions primarily between regions of DNA (cisregulation) or between DNA and another molecule such as a regulatory protein (transregulation). Both of these forms of interaction are deeply involved in the essential mechanism underlying the action of transcription factors: the promoterenhancer interaction (PEI). The PEI links together a promoter, the region encompassing the vicinity of a transcription start site of a gene and the target of the transcription factor binding, and an enhancer, a distantly positioned genomic region that acts as a facilitator of the transcription. When active, a promoter and its corresponding set of enhancers are connected with the help of activator proteins and other regulatory factors, and form a small, distinctive chromatin loop [1–3].
Promoterenhancer interactions are central to the current understanding of transcriptional regulation, and studying them requires methods which are able to capture the location, prevalence and changes of the chromatin looping. The method most directly targeted at capturing these chromatin features thus far has been chromatin conformation capture or 3C, originated almost two decades ago [4] and subsequently advanced with the advent of nextgeneration sequencing to create HiC [5]. HiC is theoretically capable of capturing all chromatin interactions between two genomic sites, however, in its classic form it is generally insensitive to PEIs due to rarely providing reliable data at high enough resolutions to capture smallscale chromatin looping [6–8]. Several approaches exist to improve this resolution, including much greater sequencing depths [9] as well as superior restriction enzymes and other technical improvements [7]. One particular method, capture HiC (CHiC), adds a sequence capture step that pares down the range of interactions to a smaller subset associated with defined genomic regions (’baits’) [10, 11]. This allows for easier capture of a particular kind of interaction and has been successfully applied to study PEIs specifically in a variant of the method called promoter capture HiC, or PCHiC [11, 12]. The single largest PCHiC study to date was conducted by Javierre and colleagues [12] on a variety of human cells belonging to the haematopoietic lineage. This study found that each particular lineage of cells had noticeable differences in the distribution of its PEIs and that many of these interactions appeared to match up with putative genetic variations linked to disease and altered gene expression. Due to its excellent coverage of a range of closely related noncancerous cell lineages the study provides an attractive target for further analysis, particularly for using computational methods to find patterns in chromatin dynamics.
Analysing HiC data is often nontrivial. Many techniques can be employed to improve the fidelity of a given dataset, from more effective experimental and data processing pipelines to postprocessing methods for finding particular features within a dataset [13–15]. Fewer analytical methods, however, focus on the dynamics of the chromatin architecture and its specificity [16]. Clustering techniques in particular may be useful in detecting functional modules of genes in HiC data. This can be observed in approaches such as ArboretumHiC, which use spectral clustering to identify groups of interactions conserved between several HiC datasets [17], or GraphTeams, where a δteams model is used to locate gene clusters in HiC data [18]. Similar techniques may be useful in detecting features in chromatin interaction landscapes that map to broader mechanisms of regulatory interaction such as transcription factories [19] or activity hubs [20]. This makes clustering a potentially valuable technique for interpreting PCHiC datasets, where functional modules made up of PEIs rarely caught in lowerresolution HiC data might be isolated. To our knowledge, however, the employment of topologybased clustering approach in the analysis of capture HiC data still has been quite limited – the approach has been used in our own previous related work [21] and for (ChIAPET network analysis) in [22].
From the topological features of PCHiC interactions analysed in [21] the most notable was the observation that PCHiC interaction networks tend to separate easily into comparatively small and welldefined connected components when we restrict the analysis to the networks in which all interactions are required to be present in several (two or more) different cell types. It was also observed that connected components of interaction networks have a tendency to remain largely unchanged when the presence of the same interactions is additionally required for a number of other (componentspecific) cell types and largely (or completely) disappear when the presence of the same interactions is additionally required for several other (componentspecific) cell types. Such a feature provides an additional indication that PCHiC interaction network components are likely related to some biological functionality and for several components manually selected for further biological validation their relation to functionally related gene modules has been shown [21]. Still, in [21] the network components were obtained by a manual adhoc procedure (by choosing a certain subset of cell types and visually exploring how the networks change when other cell types are either added or removed), leaving open the question how pronounced such component structure is, and the problem of automated identification of ’potentially interesting’ components remaining a challenge.
This behaviour was observed for the network describing PCHiC interactions for 17 different haematopoietic cell types. It would have been very interesting to check whether the same type of behaviour is characteristic for PCHiC interaction networks for different sets of cell types, however, to our knowledge no other data set suitable for such a purpose has yet become available. Due to this, we are focusing our analysis on the available PCHiC interaction networks for 17 haematopoietic cell types.
The identification of network components is related to the problem of comparison of one or more networks, which has been widely studied from various perspectives, and with particularly strong focus on biomolecular networks. For the latter, probably one of the most successful approaches has been based on graphlet sampling ([23–25]). However, in the our case comparison of two networks is not an issue (since the networks are labelled this can be done in linear time), the task is to identify potentially significant or interesting components that discriminate between 2^{k} networks assigned to each subset of k objects (cell types). The problem is quite specific and, as far as we know, has not been studied before. In slightly generalised terms it can be described as follows.
The initial data are represented by a rooted tree with graphs (networks) assigned to its nodes in such a way that graphs assigned to child nodes are (not necessarily proper) subgraphs of the graph assigned to their parent node. (In our particular case this rooted tree is a binomial tree with its vertices corresponding to all the possible 2^{k} subsets of set consisting of k cell types.) The objective is to find a set of ’characteristic’ components (subgraphs) that best differentiate between all the pairs of graphs assigned to the nodes. In principle this can be done by a straightforward twostep approach: 1) finding such characteristic set for all the pairs of nodes, and 2) combining these sets into a single set that does not contain identical or very similar components. However, such approach requires high time and space complexity; in particular, the complexity of the second step depends on both the number of nodes and sizes of networks, and for wholegenome chromatin interaction networks poses a challenge even for small number (e.g. 17, as in our case) of cell types. The alternative method that we propose does this by a onepass traversal of the tree and has significantly lower time and space requirements. The overview of the problem and the method is illustrated in Fig. 1.
The proposed approach is particularly suited for identification of connected components, but in principle could be adapted for identification of certain other characteristic topological features of the networks. It is also worth to note that whilst the networks are directed (with edges from ’baits’ to ’other ends’), the edge direction is currently not taken into account in exploring network component structure. There are two (related) main reasons for this: 1) there are different alternatives how to define directed components (the use of strongly connected components is the most obvious one, and we have also partially explored this option; however, it does not seem too promising, one of the problems being that they cover only a small part of the networks – due to the fact that all the nodes must be both ’baits’ and ’other ends’); 2) although there is limited evidence of the importance of edge directionality (e.g. statistical significance of the presence of cycles of length 2 and of biconnectivity has been observed in [21]), the role of edge directionality is still insufficiently understood to take it into account when analysing the network component structure.
The main contributions of this paper are: 1) demonstration of strong evidence of wellpronounced component structure of chromatin interaction networks and the fact that many of these components tend to be largely preserved in some of cell types and largely absent in some others; 2) a method for automatic detection of all the possible candidates for potentially biologically interesting (as measured by a scoring function assessing their significance) of such network components; 3) characterisation of the component structure of PCHiC interaction networks gained from the network analysis using this method; 4) assessment of the potential biological significance of identified network components by a ’highthroughput’ approach, whose results confirm that the components tend to be related to specific biological mechanisms/functionality.
However, whilst the limited assessment of the biological significance described here shows statistically significant relation of components to gene regulation patterns, it is very likely that the component structure is the result of several different interrelated biological processes and in this paper we do not attempt to assign to them a conclusive welldefined biological explanation. What we think is significant, is the fact that the component structure of chromatin interaction networks is sufficiently well manifested to be taken into consideration when analysing these networks. Here we propose a method that can be used for efficient automatic discovery of such components, which afterwards can be subjected to different assessment methods for their biological role and/or significance.
Methods
Data sets used for network analysis and result validation
For analysis of PCHiC interaction network topology we use a data set of longrange interactions between promoters and other regulatory elements that was generated by The Babraham Institute and University of Cambridge [12]. This data set is still largely unique because it contains genomewide data covering a representative subset of the entire haematopoietic lineage collected using a unified protocol. The data was obtained by promoter capture HiC (PCHiC) in 17 human primary haematopoietic cell types (shown in Fig. 2), and from 31253 identified promoter interaction regions across all chromosomes, a subset of highconfidence PCHiC interactions have been selected using CHiCAGO pipeline [26]. These data are available from the Open Science Framework website [12].
For the assessment of the potential biological significance of the found network components, several approaches were considered based on possibilities to perform assessments in a ’highthroughput’ manner. The options, however, are limited by the availability of datasets covering the particular 17 haematopoietic cell types. The most straightforward approaches are looking for the similarity of regulation or expression patterns of genes from the same component. Regarding the gene expression, there are few gene data sets that could be used (e.g. BLUEPRINT RNAseq data set EGAS00001000327), however, the coverage of the gene set for which there are PCHiC interaction data is rather poor. More accurate data related to the gene expression could be obtained from FANTOM5 promoter level expression atlas [27]. This data set contains transcription start site activity data obtained by CAGE [28] protocol and the available data cover genomewide information about 14 human haematopoietic cell types, 11 of these overlap with cell types for which PCHiC interaction data are available. Thus, this data set might seem to be very appropriate for biological validation, unfortunately the coverage again is quite poor – the expression data are available for approximately 18% of vertices (corresponding to ’bait’ ends) of PCHiC interaction graphs constructed from [12] data.
Regarding the gene regulation patterns, a wellsuited data set is chromatin annotations generated through ChromHMM based on ChIP data from BLUEPRINT [29]. The dataset includes information on transcription factor binding sites, CTCF binding sites, DNasesensitive sites, transcription start sites, proximal and distal cisregulatory regions, as well as the activity modes of transcription start sites and cisregulatory regions, as observed from the aforementioned annotations. The same annotations were previously used in the original publication [12] to validate the interactions between promoters and regulatory elements, but their analysis did not focus on the discovery of potential hubs of the activity or coordinated repression. The annotations are available for 9 of 17 cell types for which there are PCHiC data and vertex coverage is also quite good. For this reason we have chosen to use it here for assessment of biological significance of the identified topological features (connected network components with significance scores above a certain threshold). The data are available from Open Science Framework website [12].
Graph representation of PCHiC interaction networks
Technically the network of PCHiC interactions is described as a digraph (directed graph) G with a set of vertices V=V(G) consisting of promoters (’baits’) and detected interaction regions (’other ends’). (A vertex can also be both: a ’bait’ and an ’other end’.) For convenience, however, such vertex set V we represent simply as a set of integers {1,…,n}, where n=V.
The set of edges E=E(G) corresponds to detected interactions and edges are directed from ’baits’ to ’other ends’ (it is possible that for some vertices v_{1},v_{2}∈V both edges (v_{1},v_{2})∈E and (v_{2},v_{1})∈E). When constructed from HiC data, the set of edges E usually will depend on the selected interaction score threshold (only interactions with scores not below the threshold will be chosen for edges). For dataset analysed here we use the same threshold that was proposed in [12] – i.e. only interactions with score 5 or above are selected for edges.
By \(\mathcal {T}\) we denote the set of all the available cell types. For convenience we also assume that these cell types are indexed, i.e. \(\mathcal {T}=\{t_{1},\ldots,t_{k}\}\), where k is the number of available cell types. (For dataset in our study we have k=17.) For \(t\in \mathcal {T}\) its index is denoted by idx(t) for set \(T\subseteq \mathcal {T}\) we define idx(T)=max{idx(t)∣t∈T} – i.e. the maximal index of T elements.
For each edge e∈E there is assigned a nonempty set of labels \(T(e)\subseteq \mathcal {T}\). These labels correspond to cell types for which the scores for the interaction reached at least the threshold level. For \(T\subseteq \mathcal {T}\) by G(T) we denote a subgraph of G with vertex set V and edge set {e∈E∣T⊆T(e)}.
A connected component (or CC) of a digraph H here we simply will define as a connected component of the corresponding undirected graph \(\hat {H}\), obtained from H by ignoring edge directions (i.e. we will not require strong connectivity of components). The fact that C is a connected component of digraph H we denote by C≤_{cc}H. For \(T\subseteq \mathcal {T}\) and C≤_{cc}G(T) by G(C,T) we denote a subgraph of G(T) with set of vertices restricted to V(C) – this will be useful to discuss changes of component C when T defining it is replaced by another set T^{′}.
Algorithm for network analysis
Our aim is to find the set of all connected components \(\{C \leq _{cc} G(T) \mid T\subseteq \mathcal {T}\}\) that are ’mediumsized’ (with number of vertices n between some thresholds n_{min} and n_{max}) and have ’sufficiently interesting properties’ to merit further exploration of their potential biological significance (this is assessed by function SIGNIFICANCESCORE, a reasonably good candidate for which we are proposing below, but the function likely could be further elaborated and/or modified to improve its biological accuracy or to adapt it for assessment of different kinds of biological features).
For identification of all such connected components, we propose the algorithm FINDNETWORKCOMPONENTS (Algorithm 1) described below. The algorithm is based on BFS (Breadth First Search) of binomial tree defined by subsets of \(\mathcal {T}\) (with k+1 levels i=0…k) and all the possible i element subsets of \(\mathcal {T}\) at level i).
At each depth level i=0...k queue Q_{i} initially contains all the connected components C≤_{cc}G(T) for all \(T\subseteq \mathcal {T}\) with T=i satisfying certain pruning criteria. Q_{i} also contains components C≤_{cc}G(T^{′}) with T^{′}<i further analysis of which have been pruned earlier and which are marked as inactive. During the processing of Q_{i} components with significance score reaching threshold s or above are printed and the queue Q_{i+1} for the next level of the binomial tree is constructed. The construction of Q_{i+1} is omitted for the last depth level i=k. The algorithm prints a list of all components, together with subsets of cell types \(T\subseteq \mathcal {T}\) defining them and their significance scores. Schematically the search tree constructed by the algorithm is shown in Fig. 3.
The algorithm is called on initial graph G=G(∅) that is assumed to be based on interactions for all the chromosomes, however, since there are very few interactions between different chromosomes, technically it is more convenient to build and analyse G for each of 23 chromosomes separately. For our dataset, there seems to be a good motivation to chose n_{min}=10, the choice of n_{max} is more arbitrary, we used n_{max}=100 to limit components to a manageable size for further analysis. The value of s depends on the properties of function SIGNIFICANCESCORE; in our case by its design it was appropriate to choose s=0.
The purpose of SIGNIFICANCESCORE function is to guess/predict how ’interesting’ from the biological perspective the detected CCs could be. Since we are mostly interested in the variability of CCs for different cell types, it is natural to ask that for a C≤_{cc}G(T) with n=V(C) vertices and m=E(C) edges there is a cell type \(t^{\prime }\in \mathcal {T}T\), such that G^{′}=G(C,T∪{t^{′}}) preserves most of C edges, and also a cell type \(t^{\prime \prime }\in \mathcal {T}T\), such that G^{′′}=G(C,T∪{t^{′′}}) preserves only few of C edges. It is also natural to score higher CCs that have more options for choice of such types t^{′} and t^{′′}. Often used and well suited choice for a scoring function with such properties is (for easier readability denoting the number of edges E(H) of a graph H by e(H)):
The constants a and b should satisfy 0≤b<a≤1 and empirically were chosen as a=0.75 and b=0.25, (for the data set used it was also observed that slight variations of them do not have a significant impact on the results – see Table 1). The square root taken from the product of cardinalities of two sets does not affect the ordering of significance scores, but provides more linear distribution of their values.
Algorithm complexity
The algorithm involves traversing 2^{k} vertices corresponding to all the possible subsets \(T\subseteq \mathcal {T}\) thus its time complexity unavoidably is exponential in k. Most of the tasks involving the processing of graphs (splitting into CCs, computing C(T,E)) can be done in time O(m) (where n and m correspondingly are numbers of graph vertices and edges). Checking whether a constructed CCs C should be pruned (lines 14,15,16 of the code) can be done in time O(2^{k}V(C)).
This gives the overall algorithm time complexity O(2^{2k}n+2^{k}m). The main space requirements are for storage of two queues containing information for up to \(\binom {k}{\lfloor k/2 \rfloor } n\) graph vertices in each, thus algorithm space complexity is O(2^{k}n).
These bounds, however, are not very informative for comparatively low values of n, m and k for our particular PCHiC data set, since the actual complexity depends on the number of CCs constructed by the algorithm and the number of pruned search tree branches as well as levels at which the pruning occurs. For this dataset we have k=17 and, depending on the chromosome, n varies between 2000 and 18000 vertices and m between 3000 and 44000 edges. For these values the number of CCs is notably lower and the extent of pruning notably higher than the indicative asymptotic bounds.
In practice the current C++ implementation of the algorithm (on a single core of Xeon E51620 v3 3.50GHz processor) for processing of single chromosome data required up to 7 minutes of running time and up to 300 MB of RAM. Thus, the algorithm is quite practical for analysis of genomewide HiC interaction data for k=17 cell types, however, exponentiality in k also means 17 cell types could be already quite close to the upper limit to which the algorithm can be directly applied.
Relation with previous work and alternative approaches
The fact that PCHiC interaction network G(T) separates easily into comparatively small (with sizes up to few hundred vertices) connected components, when set of cell types T contains two or more (sufficiently distinct) haematopoietic cell types was observed by the authors in [21] by the exploration of visualised interaction networks. It was also observed that connected components have a tendency to remain largely unchanged when shared by a number of additional different (componentspecific) cell types and largely (or completely) disappear when shared by some other (component specific) cell types. A component structure of G(T) for several particular choices of the set of cell types T was explored in more detail, and few components were chosen for analysis of their biological significance. It was shown that these selected components form functionally related gene modules.
The exact component structure of G(T), however, depends on the choice of T, and one of the main contributions of this work is the algorithm for automatic detection of all connected components C≤_{cc}G(T), which are assessed as potentially biologically significant, for all the possible choices of \(T\subseteq \mathcal {T}\). The algorithm also prunes the list of components found by not searching for any subcomponents C^{′}≤_{cc}G(C,T^{′}), if component C≤_{cc}G(T) has already been output as significant for some T⊆T^{′}. The pruning, however, still does not exclude only partially overlapping components, or components of G(T) and G(T^{′}) for only partially overlapping T and T^{′}.
The direct ’automatisation’ of manual choosing of sets of cell types T that produces G(T) with welldefined component structure (which was attempted in [21]) could be achieved by a similar but simpler algorithm based on BFS of binomial tree defined by subsets of \(\mathcal {T}\) and separate analysis of the component structure of whole graphs G(T) associated with the corresponding vertices of the binomial tree. However, it is not difficult to show that the proposed analysis at the level of individual components of G(T) instead of whole graphs G(T) still outputs all the components that will be assessed as significant by SIGNIFICANCESCORE scoring function. An additional notable benefit is a significant reduction of the number of components output by the search procedure due to omitting of components that are identified as subgraphs of others.
Assessment of the biological significance
The strongly pronounced tendency of chromatin interaction networks to split into well defined medium sized connected components is clearly not a feature shared by random graphs and very likely should be explained by some biological reasons. Whilst there is also a possibility that at least partially this topological feature might be the result of limited freedom of possible conformations of chromatin 3D structure, even in this case it should likely have some impact of correlated functionality of genes contained in a particular component.
At the same time, it is very unlikely that this component structure could be attributed to a single and clearly defined biological mechanism, and these components should be regarded as good candidates for modules of genes on which to focus attention for a more thorough exploration of their potential biological significance.
In [21] we have analysed a number of selected components for enrichment with registered transcription factor proteinprotein interactions, known transcription factor binding sites, coexpressed transcription factors and binding motifs with the Enrichr web tool [30, 31]. It was found that genes contained in specific components tend to be associated with common transcription factors. Such analysis, however, is difficult to perform for a large number (few tens of thousands) of components that are identified automatically.
Here we have used another more ’highthroughput’ approach for assessment of the potential biological significance of network components. It is based on looking for relations of components to activity modes of the involved transcription start sites, proximal and distal cisregulatory regions using chromatin annotation data generated through ChromHMM based on ChIP data. The data set assigns transcription start sites (TSS) and/or regulatory regions to a number (not all) interactions in PCHiC network, which are further characterised by 4 different activity modes denoted by 0 (’dead’),1 (’active’), 2 (’poised’),3 (’Polycombrepressed’). These assignments are specific for each of cell types from \(\mathcal {T}\) and the data are provided for 9 different cell types.
To assess whether there is a tendency for network components to be associated with certain activity modes, for each component C defined by \(T\subset \mathcal {T}\) and for each cell type t∈T, for which data is available, we construct a 4tuple 〈a_{0},a_{1},a_{2},a_{3}〉. a_{i} value is equal to the number of edges e∈E(G(C,{t})) to which is assigned activity mode i, multiplied by 4tuple specific normalisation constant c, chosen to ensure that a_{0}+a_{1}+a_{2}+a_{3}=1. Thus, to each component C there is assigned a r×4 size matrix (where r depends on the number of available data entries), with rows consisting of 4tuples for these t∈T for which data are available. The largest possible value or r is 9, and we consider only components with r>1. The level of association of C with particular activity mode i can then be assessed by comparing the variance of a_{i} values within matrix assigned to C with the overall variance of a_{i} values and/or with the variance of a_{i} values for randomised data. Another option is comparing the distribution of average or maximal values of a_{i} within components with the average or maximal values for randomised data.
Results
Topological features of PCHiC networks
Since there are almost no interactions between different chromosomes in the available PCHiC dataset (from [12]), it was natural to construct and analyse interaction networks separately for each chromosome. The data set also contains very few interactions for Y chromosome, thus only interaction networks for chromosomes 1…22 and chromosome X were analysed (for technical convenience the latter here is also called ’chromosome 23’). The number of vertices in the chromosomespecific networks ranges between 2904 (chromosome 22) and 23079 (chromosome 1), the overall number of vertices in 23 interaction networks is 251209 and the overall number of edges is 723165. The number of edges to which each of 17 cell types is assigned varies between 100000 and 200000 (see Fig. 4).
The fact that PCHiC interaction networks G(T) separate easily into comparatively small connected components was already observed by the authors in [21]. For T=∅ (i.e. no requirement for edge presence in any particular cell type) the networks, however, consist of one large component containing 40−75% of G(T) vertices, the other CCs are noticeably smaller (except for chromosome 18 for which the two largest components correspondingly contain 1925 and 1178 vertices), and there are very few (2 to 14) CCs with 10 or more vertices (the choice of n_{min}=10 is somewhat arbitrary, but quite well suited to remove ’random noise’). The welldefined structure of mediumsized components start to appear when T contains 2 or more not too similar cell types – the sizes of the largest CCs tend to drop below 150 and there are about 50 (on average, the numbers are generally proportional to the size of G(T)) components with 10 or more vertices per chromosome.
For finding connected components FINDNETWORKCOMPONENTS algorithm was tested with parameters n_{min}=10,s=0 (the only natural choice this particular SIGNIFICANCESCORE function) and with different values of n_{max} and of a and b in the SIGNIFICANCESCORE function.
The value of n_{max} should not be too large (to limit components to manageable size for further analysis) but also not too small (there is a quite large number of components with 70−80 vertices and high significance scores). Increasing n_{max}, however, leads to the output of fewer components and n_{max}=100 was selected as a convenient choice from interval where small variations of n_{max} do not significantly affect the number of components found by the algorithm.
The effect of the choices of a and b on the number of components found is illustrated in Table 1 and the choice of the retained edge level thresholds a=0.75 and b=0.25 can be considered as a reasonably good compromise. An interesting seemingly counterintuitive feature is an increase of the number of components for higher a values, which is due to the pruning occurring at lower levels (as a result the algorithm outputs several partially overlapping subgraphs of a larger component that by itself fails the score). However, there is no obvious choice for the ’optimal value’ of a.
The whole data set was analysed using the parameter values n_{min}=10,n_{max}=100,a=0.75 and b=0.25. The number of components found for each chromosome range between 492 and 4107 and is shown in Fig. 5 (additionally shown are the numbers of components with less than 25 and less than 50 vertices). The distribution of the component sizes (number of edges and number of vertices) is shown in Fig. 6 (the data are shown for chromosome 6 but distributions are very similar for other chromosomes). The average edge to vertex proportion within components is slightly larger than in the whole interaction network.
A ’typical’ larger component C with a comparatively high significance score 6 from chromosome 4 is shown in Fig. 7. It is defined by the set of labels T={nB,aCD4,Neu}. At least 75% of its 80 edges are preserved in subgraphs G(C,T∪{t}) with t∈{tB,tCD8,nCD8,naCD4,tCD4,nCD4,FoeT,Ery,Mon} and at most 25% edges are preserved in subgraphs G(C,T∪{t}) with t∈{EP,Mac2,Mac1,Mac0}. As an illustrative example the subgraph G(C,T∪{EP}) is swown.
The structure of the ’component space’
From the number of components that are found by FINDNETWORKCOMPONENTS algorithm (Fig. 5) it is obvious that they must have overlapping sets of vertices. E.g. interaction graph for chromosome 1 contains 23079 vertices, however, the algorithm outputs 3066 components with at least 10 vertices each (the total number of vertices for all components is 162172).
By its design FINDNETWORKCOMPONENTS algorithm prunes the list of components found by not searching for any subcomponents C^{′}≤_{cc}G(C,T^{′}), if a component C≤_{cc}G(T) has already been output as significant for some T⊆T^{′}. This, however, does not exclude the possibility to output partially overlapping components, and from the results, it is clear that the amount of overlapping components is still significant.
The pruning also does not exclude finding components of G(T) and G(T^{′}) with the same set of vertices if there is only a partial overlap of T and T^{′}. The number of latter, however, is quite small.
To analyse the degree of overlapping between components we compared them using a simple similarity score. For two components C and C^{′} their similarity score is defined as V(C)∩V(C^{′})/V(C)∪V(C^{′}) and ranges from 0 (for nonoverlapping components) to 1 (for components with the identical set of vertices). For each of the chromosomes allagainstall comparison of components was performed and similarity scores between them have been computed. A sample distribution (for chromosome 6, however, the distributions for other chromosomes are similar) of similarity scores is shown in Fig. 8.
More informative probably is representation of component similarity by graphs (’CC graphs’) in which components correspond to vertices and edges connect pairs of components with similarity score above some given threshold. For analysis of such ’CC graphs’ it again is useful to look at their component structure. A ’good feature’ of these graphs is that they split into comparatively (i.e. close to complete graphs) components that remain mutually isolated. This can be considered as another indirect confirmation that component structure is characteristic to PCHiC interaction networks and not the result of specific technique of network analysis. The number of components in ’CC graphs’ and their sizes depend on the similarity threshold used. Table 2 shows number of components of ’CC graphs’ for chromosome 6, their maximal and average sizes as well as density (edge density of components with n vertices and m edges is defined as 2m/n(n−1) and shows how close these components are to complete graphs) for similarity thresholds from 0.00 to 0.95. The average sizes and average densities are computed only for components with 4 or more vertices. The data for all 23 chromosomes and similarity threshold 0.75 are shown in Table 3.
Part of ’CC graph’ for chromosome 6 is visualised in Fig. 9 (the largest part of the graph’s 2355 vertices, however, are contained within components of 1 to 4 vertices, which are not shown here). This figure shows a typical component structure of ’CC graphs’ regardless of the used similarity thresholds, although for the lower thresholds there are fewer components and average and maximal component sizes increase.
At present this description about the structure of ’component space’ is provided only as being potentially useful for interpretation of the results produced by FINDNETWORKCOMPONENTS algorithm. Nevertheless, it is also clear, that reducing the number of components by including only representatives from sets of very similar ones (and probably excluding some that are of little interest – e.g. quite typical ’star like’ structures) would facilitate analysis of their biological roles. However, this unlikely can be done on the basis of similarity scores alone. A second semisupervised processing stage for selecting ’representative set’ from the components directly output by the algorithm remains an interesting option to explore.
Assessment of the biological significance of network components
The assessment of the potential biological significance of the components was performed using gene regulation annotations, and the results show that on average the genes from the same component have similar regulation patterns, thus in principle confirming that components of PCHiC interaction networks are related to specific biological functionality and/or biological mechanisms of their formation. A brief summary is given by Fig. 10 showing variance of regulation activity modes within network components, which for most of the components are below the overall variance values. There is also a strong tendency for each component being associated with this componentspecific activity mode. This effect in comparison to the distribution for randomised data is shown in Fig. 11. The exact calculation of statistical significance of this effect is difficult due to complex dependencies between the component edges. We computed the approximate pvalues with the Wilcoxon test [32] (using its standard implementation in R language; the test itself can be considered to be the most appropriate for the data that are not normally distributed). For all the chromosomes the obtained pvalues were 2.2×10^{−16} (i.e. ’almost zero’) when comparing activity distributions between our components and randomised data. Whilst such estimate is not totally accurate, it is still a very convincing indication that the statistical significance is high.
The activity assessments provided for the predicted transcription start sites and enhancers in the whole PCHiC dataset were essentially dominated by two major groups: active regions (activity "1") and dead zones (activity "0"), both making up approximately half of all activities observed. Poised (activity "2") and Polycombrepressed (activity "3") sites were comparatively much rarer, together making up no more than 10−15% of all activities in every chromosome except for chromosome 17, where poised activity was predicted in 22% of sites in the whole data set. When compared to this overall background, our components showed mild but noticeable differences in the activity. Foremost of these was an enrichment in predicted dead zones, with a mean 5% increase across all chromosomes, the highest enrichment of which (13%) was observed in chromosome 13. Conversely, active sites seemed comparatively diminished in our components, showing a largely consistent decrease in prevalence across most chromosomes. Polycombrepressed sites were mostly enriched as well but remained in the 1−3% range of prevalence.
When considering distributions of activities within components, we found that approximately two thirds of the components showed a strong primary activity predicted for 60% of sites or more. Much like in the overall distribution, most of these are dominated by either predicted active sites or dead zones. There are also approximately 100 components showing a majority of poised activity, but only three dominated by Polycomb repression. Moreover, almost all of these components show noticeably low differences in the activity types detected between the tissue types that they primarily belong to, with variances between tissue types generally well below those observed in random permutations of the same data (Fig. 10). Altogether these results point toward shared properties within individual components, although no unified significance that applies to every component can be isolated through our method.
Discussion
Our initial goal in setting the criteria for suitable components was to find dynamic structural elements in chromatin corresponding to tissuespecific modules of promoterenhancer interactions. Our results instead show that our components, and also the PCHiC data they stem from, connect a wider variety of structures, including most notably a wide range of components that connect chromatin ’dead zones’ with an overall quiescent profile of histone marks [33, 34]. These components occur in roughly equal proportion to ones that are dominated by markers of activity, and in fact, are more frequent in our filtered component set compared to the full set of annotated edges. As such, an important question to ask would be how to differentiate such components in our data, if it is possible. Furthermore, it may be important to also consider other components previously filtered out in our selection process and to analyse what each of them may contain.
More elaboration on the content and selectivity of the components could potentially be obtained in several ways. Firstly, with further data from a wider variety of independent datasets such as the FANTOM5 promoterlevel expression atlas [27]. Of particular note may be ATACseq and DNase accessibility studies, as DNA accessibility is an obligatory prerequisite of enhancer function, whereas particular histone marks do not necessarily enable or preclude such function in vivo [35]. Additionally, even with the data we have currently gathered, it may be possible to find further topological properties of components such as particular sizes or modes of connectivity which help differentiate between the components already found. As we have demonstrated in our previous work, metrics based on graph topology can be used to differentiate between broad categories of tissue types [21], and so may be useful in finding out the general properties of highly active and spatially compact gene clusters.
Our analysis could also benefit from the reexamination of the components we have filtered out in the present study. The current criteria stipulate that each component must be specific to a small number of tissue types, but still retain most of its interactions in at least one more tissue. Given that these criteria eliminated the majority of the connected components found in the original dataset, it is likely that we could glean some insight from both nonspecific components found in most or all tissue types studied, and also highly specific components delineating the most variable elements of dynamic chromatin architecture. In fact, understanding the contribution of the latter category to the regulatory landscape of blood cells may be the logical next step in further work, with nonspecific chromatin architecture serving as a useful frame of comparison.
Regarding the algorithm FINDNETWORKCOMPONENTS for identification of connected components of potential biological significance the part that likely can be further elaborated/improved is SIGNIFICANCESCORE function. In current version significance scores do not depend on the size of connected components and high scores are assigned also for comparatively small and simple components that (depending on the biological questions further asked) might not always be of particular interest. A better insight into component topology and its relation to biological functionality, however, is needed for this.
As already mentioned, reducing the number of components would facilitate analysis of their biological roles and a second semisupervised processing stage for selecting ’representative set’ from the components directly output by the algorithm remains an interesting option to explore.
Conclusions
In this paper we have presented a novel algorithm for analysis of chromatin interaction networks with a goal to identify characteristic topological features of interaction graphs and to ascertain their potential significance in chromatin architecture. The algorithm provides automatic identification of all connected components with significance score above the given threshold that can be potentially related to specific biological role or function. The fact that chromatin interaction networks tend to separate easily into welldefined connected components to which it is possible to assign certain biological functionality was previously observed by the authors [21]. However, identification of such components was only possible with manual adhoc methods, with which exploration of the whole component space was infeasible.
We have applied the developed FINDNETWORKCOMPONENTS algorithm to analysis of PCHiC interaction networks of 17 different haematopoietic cell types based on dataset by [12]. This provided strong evidence that component structure for these PCHiC interaction networks is quite pronounced and also allowed to obtain some characterisation of this component structure. We have also made assessment of potential biological significance of the found network components by analysing regulation patterns of genes contained within components and the results confirmed that on average genes from the same component have similar regulation patterns. At the same time it is very likely that the component structure is the result of number of different interrelated biological processes, and by no means we attempt to claim that we have assigned to them a unique welldefined biological explanation. What we think is significant, is the fact that component structure of PCHiC interaction networks is sufficiently well manifested to be taken into consideration when analysing such networks.
The developed algorithm can be adapted for exploration of similar data sets of PCHiC interactions (or chromatin interactions obtained by other technology) that includes information for sufficiently large number of different cell types. The analysis results obtained on another data set would provide significant new insights whether topological structure of the chromatin interaction networks is similar for different sets of cell types, and whether there are similar associations of structural components with specific biological functionality. Unfortunately, to our knowledge no other data set suitable for such a purpose as yet have become available, however, emergence of such data sets in near future is very likely. In particular, there are already available several genomewide chromatin interaction data sets that cover few (up to 3) different cell types (e.g. [22]). Whilst data about only 3 cell types are really insufficient for component structure exploration (and the structure of ChIAPET networks studied in [22] is somewhat different), the study provides an additional support to the hypothesis of importance of interaction network component structure. The authors of [22] have provided characterisation of biologically significant topological features of the networks in terms of graphlets (small subgraphs), although, in contrast to our approach, these have been derived from the known biological features, whilst our emphasis is on potential discovery of new biological relations from analysis of network topology.
More problematic, but still possible, is adaptation of the method for other types of cell (or tissue) typespecific networks. This could be quite feasible for gene regulatory networks (although instead of connected components, these likely will need to be analysed in terms of more complex topological structures); the main limitation, however, is the fact that currently the tissuespecific information on gene regulation is mostly provided by the chromatin interaction data. Still, a possible application could be analysis of gene regulatory networks of different species based on the known homologies between the genes. Although such task will be more complicated and it is difficult to predict how interesting and/or useful the results might be.
The developed software for automatic identification of components in PCHiC interaction networks (C++ code and compiled Windows and Linux binaries) is publicly available at GitHub repository: https://github.com/IMCSBioinformatics/ PCHiCNetworkExplorer. The repository contains also data files describing analysed PCHiC interaction networks used for experiments as well as analysis results – list of identified components with assigned scores of their anticipated biological significance. The software also includes a JavaScript based webbased browser for visualisation and exploration of components of PCHiC interaction networks.
Availability of data and materials
The developed software for automatic identification of components in PCHiC interaction network (C++ code and compiled Windows and Linux binaries) is publicly available on GPL license at GitHub repository: https://github.com/IMCSBioinformatics/PCHiCNetworkExplorer. The repository contains also data files describing analysed PCHiC interaction networks used for experiments as well as analysis results – list of identified components with assigned scores of their anticipated biological significance.
The software also includes a JavaScript based web browser for visualisation and exploration of components of PCHiC interaction networks. This is a modified version of the browser that was previously made available as a supplement to an earlier publication of the authors [21].
Abbreviations
 3C:

Chromatin conformation capture
 aCD4:

Activated total CD4+ T lymphocytes
 ATACseq:

Assay for transposaseaccessible chromatin using sequencing
 BFS:

Breadth first search
 CAGE:

Cap analysis gene expression
 CC:

Connected component
 CHiC:

Capture HiC
 ChIAPET:

Chromatin interaction analysis by pairedend tag sequencing
 CHiCAGO:

Capture HiC analysis of genomic organization
 ChIP:

Chromatin immunoprecipitation
 CTCF:

CCCTCbinding factor
 EP:

Endothelial precursors
 Ery:

Erythroblasts
 FoeT:

Fetal thymus
 Mac0:

Macrophages M0 (nonactivated macrophages)
 Mac1:

Macrophages M1 (inflammatory macrophages)
 Mac2:

Macrophages M2 (alternatively activated macrophages)
 MK:

Megakaryocytes
 Mon:

Monocytes
 naCD4:

Nonactivated total CD4+ T lymphocytes
 nB:

Naive B lymphocytes
 nCD4:

Naive CD4+ T lymphocytes
 nCD8:

Naive CD8+ T lymphocytes
 Neu:

Neutrophils
 PCHiC:

Promoter capture HiC
 PEI:

Promoterenhancer interaction
 tB:

Total B lymphocytes
 tCD4:

Total CD4+ T lymphocytes
 tCD8:

Total CD8+ T lymphocytes
 TSS:

Transcription start site
References
Mora A, Sandve GK, et al.In the loop: promoterenhancer interactions and bioinformatics. Brief Bioinform. 2016; 17(6):980–95.
Matharu N, Ahituv N. Minor loops in major folds: Enhancerpromoter looping, chromatin restructuring, and their association with transcriptional regulation and disease. PLoS Genet. 2015; 11(12):1–14.
Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties to genomewide predictions. Nat Rev Genet. 2014; 15:272–86.
Dekker J, Rippe K, et al.Capturing chromosome conformation. Science. 2002; 295(5558):1306–11.
LiebermanAiden E, van Berkum E, et al.Comprehensive mapping of longrange interactions reveals folding principles of the human genome. Science. 2009; 326(5950):289–93.
Schmitt AD, Hu M, Ren B. Genomewide mapping and analysis of chromosome architecture. Nat Rev Mol Cell Biol. 2016; 17:743–55.
Belaghzal H, Dekker J, Gibcus JH. HiC 2.0: An optimized hic procedure for highresolution genomewide mapping of chromosome conformation. Methods. 2017; 123:56–65.
Mishra A, Hawkins RD. Threedimensional genome architecture and emerging technologies: Looping in disease. Genome Med. 2017; 9(1):1–14.
Rao SSP, Huntley MH, et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014; 159(7):1665–80.
Dryden NH, Broome LR, et al.Unbiased analysis of potential targets of breast cancer susceptibility loci by capture HiC. Genome Res. 2014; 24(11):1854–68.
Mifsud B, TavaresCadete F, et al.Mapping longrange promoter contacts in human cells with highresolution capture HiC. Nat Genet. 2015; 47:598–606.
Javierre BM, Burren OS, et al.Lineagespecific genome architecture links enhancers and noncoding disease variants to target gene promoters. Cell. 2016; 167(5):1369–84.
Lajoie BR, Dekker J, Kaplan N. The hitchhiker’s guide to HiC analysis: Practical guidelines. Methods. 2016; 72:65–75.
Forcato M, Nicoletti C, et al.Comparison of computational methods for HiC data analysis. Nat Methods. 2017; 14:679–85.
Golloshi R, Sanders JT, McCord RP. Iteratively improving HiC experiments one step at a time. Methods. 2018; 142:47–58.
Chasman D, Roy S. Inference of cell type specific regulatory networks on mammalian lineages. Curr Opin Syst Biol. 2017; 2:130–9.
Siahpirani AF, Ay F, Roy S. A multitask graphclustering approach for chromosome conformation capture data sets identifies conserved modules of chromosomal interactions. Genome Biol. 2016; 17(114). https://doi.org/10.1186/s1305901609628.
Schulz T, Stoye J, Doerr D. GraphTeams: a method for discovering spatial gene clusters in HiC sequencing data. BMC Genom. 2018; 19(Suppl 5):308.
Schoenfelder S, Clay I, Fraser P. The transcriptional interactome: Gene expression in 3D. Curr Opin Genet Dev. 2010; 20(2):127–33.
Phanstiel DH, Van Bortle K, et al.Static and dynamic DNA loops form AP1bound activation hubs during macrophage development. Mol Cell. 2017; 67(6):1037–48.
Lace L, Melkus G, Rucevskis R, Celms E, Cerans K, Kikusts P, Opmanis M, Rituma D, Viksna J. Graphbased characterisations of cell types and functionally related modules in promoter capture HiC data. In: Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies, vol. 3: BIOINFORMATICS: 2019. p. 78–89. https://doi.org/10.5220/0007390800780089.
Thibodeau A, Marques EL, et al.Chromatin interaction networks revealed unique connectivity patterns of broad H3K4me3 domains and super enhancers in 3D chromatin. Sci Rep. 2017; 7(14466). https://doi.org/10.1038/s41598017143897.
Yaveroglu ON, Milenkovic T, Przulj N. Proper evaluation of alignmentfree network comparison methods. Bioinformatics. 2015; 31(16):2697–704.
Przulj N, MalodDognin N. Network analytics in the age of big data. Science. 2016; 353(6295):123–4.
Sarajlic A, MalodDognin N, et al.Graphletbased characterization of directed networks. Sci Rep. 2016; 6(35098). https://doi.org/10.1038/srep35098.
Cairns J, FreirePritchett P, et al.CHiCAGO: robust detection of DNA looping interactions in capture HiC data. Genome Biol. 2016; 17(127). https://doi.org/10.1186/s1305901609922.
Lizio M, Harshbarger J, et al.Gateways to the FANTOM5 promoter level mammalian expression atlas. Genome Biol. 2015; 16(22). https://doi.org/10.1186/s1305901405606.
Takahashi H, Sachiko K, et al.CAGE  cap analysis gene expression: a protocol for the detection of promoter and transcriptional networks. Methods Mol Biol. 2012; 786:181–200.
Stunnenberg HG, et al.The international human epigenome consortium: A blueprint for scientific collaboration and discovery. Cell. 2016; 167:1145–9.
Chen EY, Tan CM, et al.Enrichr: interactive and collaborative html5 gene list enrichment analysis tool. BMC Bioinformatics. 2013; 14(128). https://doi.org/10.1186/1471210514128.
Kuleshow MV, Jones MR, et al.Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016; 44:90–7.
Wilcoxon F. Individual comparisons by ranking methods. Biom Bull. 1945; 1(6):80–3.
Ernst J, Kellis M. Chromatinstate discovery and genome annotation with ChromHMM. Nat Protoc. 2017; 12(12):2478–92.
Ernst J, Kheradpour P, et al.Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011; 473(7345). https://doi.org/10.1038/nature09906.
Catarino RR, Stark A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation. Genes Dev. 2018; 32(34):202–23.
Acknowledgements
Not applicable.
About this supplement
This article has been published as part of BMC Bioinformatics Volume 20 Supplement 23, 2019: Proceedings of the Joint International GIW & ABACBS2019 Conference: bioinformatics. The full contents of the supplement are available online at https://bmcbioinformatics.biomedcentral.com/articles/supplements/volume20supplement23.
Funding
The research and publication costs were funded by European Regional Development Fund project ”Graph based modelling and analysis methods for systems biology” (ERDF project 1.1.1.1/16/A/135).
Author information
Authors and Affiliations
Contributions
All authors have contributed to the presented research and to the preparation of the paper. JV, KC and KF developed the overall methodology and mathematical framework. GM developed methods for biological validation of the results and provided biological interpretation of validation outcomes. PK, DR and EC contributed to implementation of PCHiC network analysis algorithm and running computational experiments. LL and MO contributed to statistical analysis of the results. PR and PK developed the tools for data visualisation. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Viksna, J., Melkus, G., Celms, E. et al. Topological structure analysis of chromatin interaction networks. BMC Bioinformatics 20 (Suppl 23), 618 (2019). https://doi.org/10.1186/s128590193237z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s128590193237z
Keywords
 Chromatin interaction networks
 Graph topology
 Cell type specificity
 Functionally related modules