Previous studies have established a strong consensus that stringent regulation of CCN2 expression is fundamental in development and beyond, exemplified by pathology that results when this control is lost (Ivkovic et al. 2003). Thus far, study of the regulation of CCN2 transcription has focused on the upstream locus in close proximity to the coding sequence, in addition to within the introns and 3’ end of the gene. The aforementioned 160kb BAC sequence did not demonstrate specific cis-acting sequences with capacity to drive tissue specific expression of CCN2 (Gong et al. 2003). In this study, we investigated the 5’ end of the CCN2 sequence and have identified four cis-acting regulatory regions upstream of the coding sequence within the murine genome. Each of these enhancers was able to drive β-galactosidase reporter gene expression in a tissue-specific manner, primarily within cartilaginous, osseous and vascular cells; partially fulfilling the pattern of endogenous CCN2 expression at this time-point (Friedrichsen et al. 2003; Ivkovic et al. 2003).
A 2.3kb sequence located approximately -100kb from the CCN2 TSS drove transgene expression within the dermal microvasculature alone, with no activity observed in any other endothelial cell population. This pattern was similar to that of vascular endothelial cadherin, which has previously been shown to be present within developing dermal microvasculature; although this gene is also expressed more widely within the endothelium of soft tissue (Monvoisin et al. 2006). This -100kb enhancer is therefore utilised to drive CCN2 expression in a highly cell-specific manner. Understanding of the time-frame in which this enhancer is active was aided by the fact that a smaller fragment of approximately 1.7kb within this region has been previously screened for enhancer activity as part of the VISTA Enhancer Browser and found to be non-functional at E11.5; enhancer activity is therefore induced between E11.5 and E15.5 (Visel et al. 2007). Microvasculature based reporter gene expression has previously been described within the Tg(Ctgf-eGFP)FX156Gsat model and transgenic mice harbouring a sequence spanning from -4kb to the CCN2 TSS (Huang et al. 2010; Hall-Glenn et al. 2012). However, these constructs gave rise to a wider range of vascular elements than the -100kb enhancer. We speculate that the -100 enhancer could provide a compensatory mechanism within the endogenous system, ensuring that disruption of one enhancer’s function does not result in perturbation of CCN2 transcription in specific cell type (Perry et al. 2011; Cannavò et al. 2016).
Overlap in transgene expression pattern only accounted for a small proportion of the total activity of the enhancers, with the most substantial transgene activity observed within markedly differing cell types. The overlap in transgene activity occurred within articular chondrocyte cells in the -135kb and -198kb enhancers. Both enhancers were active within elbow and knee synovial regions, yet the -198kb enhancer demonstrated additional function close to the articular surface of hip and shoulder joints. There is some variation in the cellular signalling pathways and transcription factors that dictate the formation of different joints, for example between the knee and elbow, which could account for discretion in the joints in which the enhancers were active (Pazin et al. 2012). Limb and joint development occur in a proximal to distal manner and that there was no staining observed in the stylopod joints of the shoulder and knee for the -135kb enhancer. We therefore speculate that activity of this enhancer may have been greater in earlier time point; for example at E11.5 or E13.5, with diminishing activity by E15.5.
During embryonic development, the importance of stringent CCN2 expression within cartilaginous tissue is emphasised by the fact that three of the four enhancer regions identified here; in addition to the previously reported BAC and -4kb models (Huang et al. 2010; Hall-Glenn and Lyons 2011) exhibited transgene activity in chondrocytes. Moreover,
the extent of CCN2 expression within endochondral cartilage has been associated with differentiation state of the chondrocytes, mainly within the more mature hypertrophic chondrocytes (Friedrichsen et al. 2003; Ivkovic et al. 2003). This pattern is concomitant with that of the transgene expression driven by the -198kb enhancer where clear stratification of β-galactosidase activity was in line with the differentiation state of the chondrocytes. The proliferative chondrocytes within the resting zone were negative for X-gal staining, whereas the columnar, elongated hypertrophic chondrocytes proximal to the primary ossification centre exhibited strong transgene activity. This suggests perhaps that enhancer activity is directed primarily by transcription factors specific to this stage in endochondral ossification; such as Runx2. We have shown in supplementary Figure S3 the possible location of this binding site, along with other potential factors that can drive this expression; however more study is required to determine direct interaction. CCN2 is also expressed by hypertrophic chondrocytes postnatally within secondary ossification centres; further study could also seek to understand whether the -198kb enhancer functions at this stage or solely during embryonic endochondral ossification (Oka et al. 2007).
The role of CCN2 in ossification is not limited to the cartilaginous anlagen during endochondral ossification. Indeed, osteoblast proliferation is increased in vitro in the presence of CCN2 and expression of the gene in osteoblasts has also been observed in vivo during bone growth and remodelling (Safadi et al. 2003). The -229kb enhancer exhibited the most potent activity within osseous tissue in the primary ossification centres of the scapula and femur. This bone-specific function was most distinguishable in the mandibular tissue, with strong X-gal staining of the osseous tissue and no staining of the chondrocytes within the Meckel’s cartilage; a tissue that endogenously expresses CCN2 at this time point (Shimo et al. 2004). The strongest utilisation of this enhancer occurred within the periosteal tissue and early cortical bone. Knockout of CCN2 leads to reduced mineralisation of cortical bone at E15.5, therefore implicating CCN2 in bone collar formation (Kawaki et al. 2008). This is supported by previous study that has found CCN2 mRNA localised within periphery of early osseous tissue during embryonic development (Friedrichsen et al. 2003). Moreover, the localisation of this transgene expression tallied with the endogenous patterns of osteoblastic lineage markers Collagen type I and Osterix (Maes et al. 2010).
Although a further mouse candidate enhancer region was located -254kb from the TSS, this did not drive transcription of the transgene at E15.5 (Figure 1B). This was surprising given that the -254kb region contained the largest peaks for enhancer associated traits at E14.5 on the ENCODE browser (Figure 1A). This finding therefore highlights issues with the assumption that in silico annotation tallies with in vivo function (Dogan et al. 2015)
The specificity in the localisation of transgene expression and therefore utilisation of each of the enhancers described here underlines the importance of these regions in determining niche gene expression during development. Enhancers are increasingly being implicated as key mediators of cell fate determination, which is conferred by plasticity in chromatin state throughout the course of development (Heintzman et al. 2009; Zhu et al. 2013; Huang et al. 2016). CCN2 has previously been associated with tissues undergoing differentiation and transition of cell type; as occurs in development of cartilage from mesenchymal precursor (Friedrichsen et al. 2003). This lends credence to the notion that the enhancers identified in the current study contribute to cell lineage-determination through stratification of CCN2 transcription and therefore protein function. However, further study is required to elucidate the specific temporospatial context in which each enhancer functions. For example, assessment of enhancer function within adulthood is a critical aspect of understanding whether function is limited to during embryonic development, or whether it prevails to drive the postnatal transcription of CCN2. Furthermore, the enhancers could also function in a latent manner, with environmental stimuli triggering utilisation of an enhancer in order to drive expression of CCN2 (Nord et al. 2013). The transgene expression patterns observed in the current study are a reflection of the integration of activity from multiple signalling pathways and therefore transcription factors in a cell lineage-specific manner (Kieffer-Kwon et al. 2013; Huang et al. 2016). An important step in future study will be the identification of the key transcription factor consensus binding motifs that underpin the function of each enhancer. For this reason, we have included supplementary Figures S1-S5 which depict possible binding sites for known transcription factors which have high homology across species that can be used in mutation experiments. This includes predictions of sites within the BAC sequence previously used (S5) which may help identify the shorter elements that drive activity of transgene in vivo. The combination of in vivo study and mutational analysis would represent a powerful tool than assessment of chromatin state in enabling refining understanding of the context in which each enhancer is utilised (Dogan et al. 2015).
In summary, the expression patterns observed in this study partially recapitulate that of endogenous CCN2 during embryonic development, indicating that multiple enhancer regions are responsible for the endogenous transcription of CCN2 (Figure 6). This therefore suggests that the region of 300kb upstream of CCN2 has capacity to function as a topologically associated domain, containing enhancer regions whose composite function facilitates chromatin looping and the induction of CCN2 transcription within several tissues in a cell-specific manner at E15.5; a potential mechanism for which is shown in Figure 6 (Dixon et al. 2012). Whilst these findings give an insight into the capacity of non-coding elements to regulate the expression of CCN2, further study is required to better characterise these regions and the intricate mechanisms that dictate their function; including knocking out using CRISPR technology. Future examination of enhancer function and sequence could aid in the understanding and amelioration of diseases that involve CCN2 function as there is a strong consensus that perturbation of topological associated domains and more specifically enhancer activation contributes to pathology (Lupiáñez et al. 2015; Murakawa et al. 2016). Thus, pathological activity of the enhancers described here could contribute to aberrant expression of CCN2 in disease; for example, in osteoarthritis, especially given the prevalence of chondrocyte based enhancer function.
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Figure 1: Multiple enhancers upstream of CCN2 function at E15.5. In silico visualisation of the 300kb region upstream of CCN2 using the ENCODE browser (A) allowed the identification of candidate enhancers. Regions located -229kb (C), -198kb (D), -135kb (E) and -100kb (F) from the CCN2 TSS each exhibited capacity to drive lacZ reporter gene expression at E15.5. A predicted enhancer located -254kb from the TSS did not show any transgene activity (B).
Figure 2: -100kb upstream sequence of CCN2 gene drives transgene activity within microvasculature. Larger blood vessels were not stained (arrowheads B and C) contrasting the punctate staining in the surrounding area. Transgene localisation was concomitant with that of microvasculature and capillary vessel endothelium, with erythrocytes visible within blue branched structure (F, black arrowheads). This pattern was observed globally in a highly superficial manner such as in the frontal portion of the craniofacial region (E and F) and primordial dermis surrounding the fore limb (G) and spine (I). Staining was not observed within other populations of endothelial cells, such as in the growth plate of endochondral tissue. There was an absence of staining in any other tissue type including cartilaginous tissue of the chondrocranium (D and E), wrist (H), intervertebral disc (I), ribs (J) and femur (K). Early osseous tissue within these regions was also negative for transgene expression (Bar=100µm)
Figure 3: An enhancer at -135kb upstream of CCN2 drives transgene activity within articular chondrocytes. Strong transgene expression was observed within the proximity of synovial joints (more specifically the elbow (C), wrist (D), femoral aspect of the knee (F) ankle (G) and hind limb paw (H)). Hypertrophic chondrocytes, such as in the vicinity of the primary ossification centres of radius and ulna (C) in addition to the hip (E) and femur (F) were not stained. β-galactosidase activity was absent within all other tissues including the chondrocranium (A) and ribs (B) (Bar=100µm)
Figure 4: An enhancer -198kb upstream of CCN2 drives transgene activity within tissue of mesenchymal origin.
Strong transgene expression was observed within dermal mesenchyme globally including within the dorsal (A and D) and craniofacial (C) regions. Further fibroblastic activity was also observed within the tendons of the hind paw (B). Hypertrophic chondrocytes stained strongly in a global manner including within the basioccipital bone and atlas (E), scapula (G), humerus (H) and hind limb (K). Staining within other types of chondrocytes was limited, with additional activity within articular chondrocytes such as in the hip (J, black arrowhead). Punctate staining was observed within the chondrocranium (C and F) whereas the vertebrae (L) and intervertebral disc (black arrowhead) were negative for transgene activity. There was however, some staining within osseous tissue including within the primary ossification centre within the scapula (H, os), humerus (H, os) and iliac bone (I), this was not present in the ossified region of the orbital plate of the frontal bone (C, black arrowhead) (Bar=100µm)
Figure 5: An enhancer -229kb upstream of CCN2 drives transgene activity within mesenchyme derived tissue. Whole mount clearing of X-gal stained embryos (A) revealed strong staining predominantly within tissue subject to ossification. Marked? staining was observed within periosteal tissue within the frontal and parietal bones (B and C) in addition to the scapula (E), ulna (H), vertebrae (K), ribs (L) and hind limb (M and N). Within the mandibular region (D), osseous tissue stained strongly (ma) whereas the Meckel’s cartilage was negative (mc). Staining also occurred within cartilaginous tissue within the chondrocranium (F, G) and annulus fibrosus of intervertebral disc (P, black arrowhead). Transgene activity was also observed within the connective tissue within the dorsal dermis and intercostal regions (J and L respectively) (Bar=100µm)
Figure 6: A potential mechanism through which CCN2 transcription is regulated by multiple enhancer regions in a cell type-specific manner. Open chromatin with permissive epigenetic modification within an enhancer (such as -198kb and -229kb) is bound by transcription factors that are cell type-specific, resulting in looping of chromatin.
Further protein-protein interaction allows the assembly of the transcription initiation complex at the CCN2 proximal promoter region, resulting in transcription in a temporospatial manner; such as within dermal fibroblasts at E15.5. The function of other enhancers that may be active at this time-point within other cell-types is prevented by repressive chromatin state (such as with the -100kb and -135kb regions). Further regulatory elements within the promoter region and up to -57kb upstream, as described in previous studies, may also contribute to this refined gene expression.