Hexavalent Chromium Disrupts Chromatin Architecture

Abstract

Accessibility of DNA elements and the orchestration of spatiotemporal chromatin-chromatin interactions are critical mechanisms in the regulation of gene transcription. Thus, in an ever-changing milieu, cells mount an adaptive response to environmental stimuli by modulating gene expression that is orchestrated by coordinated changes in chromatin architecture. Correspondingly, agents that alter chromatin structure directly impact transcriptional programs in cells. Heavy metals, including hexavalent chromium (Cr(VI)), are of special interest because of their ability to interact directly with cellular protein, DNA and other macromolecules, resulting in general damage or altered function. In this review we highlight the chromium-mediated mechanisms that promote disruption of chromatin architecture and how these processes are integral to its carcinogenic properties. Emerging evidence shows that Cr(VI) targets nucleosomal architecture in euchromatin, particularly in genomic locations flanking binding sites of the essential transcription factors CTCF and AP1. Ultimately, these changes contribute to an altered chromatin state in critical gene regulatory regions, which disrupts gene transcription in functionally relevant biological processes.

Introduction

Chromium is an abundant transition metal that is most commonly found in the environment as one of three stable valences, as either trivalent (Cr(III)), hexavalent (Cr(VI)) or metallic (Cr(0). While Cr(III) is the most common form that occurs naturally, the origins of Cr(VI) compounds can largely be attributed to anthropogenic sources due to the industrial use of chromium products in stainless-steel production, electroplating, leather tanning, textile and pigment production, among others[1]. Trivalent chromium can be harmful in acute exposures, however the hexavalent form is considered to be substantially more toxic on account of its increased ability to cross cellular membranes. On the other hand, Cr(III) relies on passive diffusion to permeate the cell membrane, substantially limiting its accumulation within cells. While Cr(VI) is largely reduced in the extracellular environment to Cr(III), molecules that escape the initial detoxification process are rapidly facilitated into the cell through nonspecific sulfate/phosphate anion transporters and subsequently reduced to Cr(III) by ascorbate, glutathione, and to a lesser extent cysteine, through one- or two- step reductions (depending on the reducing agent)[2]–[6].

Consequently, studies investigating the toxicity of chromium largely focus on the hexavalent species. Specifically, Cr(VI) has been well-established as a respiratory carcinogen through multiple epidemiological and animal studies, with an emphasis on occupational settings where chromate workers are primarily exposed through inhalation and dermal contact. However, the most common route of exposure for the general population is the ingestion of drinking water contaminated with low levels Cr(VI) which may be the result of improper disposal methods of chromium waste, pollution, and natural sources. A recent study by Tan et al. has reported that interactions between cast iron pipes and chlorine disinfectants in drinking water distribution systems can promote the formation of Cr(VI)[7]. While evidence suggesting ingestion is associated with increased cancer incidence exists, the quantification of risk in the general population is not as clear. Despite this, current models of Cr(VI) uptake and intracellular metabolism suggest that carcinogenesis is inextricably linked to the molecular properties of chromium and further understanding the mechanisms through which chromium promotes carcinogenesis is important for improving the health and safety of the population. Continued advances in our understanding of the genome highlight the critical role chromatin organization plays in the function of the cell. Based on current evidence, chromium interferes with the structure and function at multiple levels of chromatin compaction. By damaging DNA directly, through the formation of DNA adducts and protein-DNA crosslinks, Cr(VI) evokes a DNA damage response that requires extensive chromatin remodeling to accommodate the repair machinery[8], [9]. At the nucleosome level, emerging evidence is showing that Cr(VI) alters chromatin accessibility by changing the nucleosomal occupancy and positional shifts[10]. Importantly, these changes occur at CTCF and AP1 binding sites, supporting a mechanism where Cr(VI) alters the DNA binding capacities of critical transcription factors, leading to an altered chromatin state. Ultimately, Cr(VI) targets active gene regulatory regions, disrupting gene transcription in biologically relevant cellular processes.

Chromatin architecture and mechanisms of chromium-mediated disruption

Cr(VI) damages DNA through direct and indirect mechanisms

The reduction of Cr(VI) in the body can be protective or toxic depending on where the reaction occurs. On one hand, reduction in the extracellular environment serves as a primary method of detoxification and largely prevents the uptake of excess Cr(III) in cells. On the other, similar processes act on Cr(VI) that is rapidly taken up inside cells and results in the production of free radicals, reactive intermediate metabolites, and accumulation of Cr(III)(Figure 1). While Cr(VI) does not directly interact with DNA, Cr(III) is capable of forming binary (Cr-DNA) and ternary (ligand-Cr-DNA) adducts. Therefore, it is thought that chromium causes DNA damage through two broad sets of processes; induction of oxidative stress and formation of chromium-DNA adducts.

Figure 1:

The biological fate of hexavalent chromium in mammalian cells. Cr(VI) is largely reduced to Cr(III) extracellularly, effectively limiting uptake of Cr(III) by cells and serving as a detoxification mechanism. The remaining Cr(VI), that escapes the extracellular reduction process mimics sulfate or phosphate ions and is transported by sulfate anion transporters into the cell. Once in the cytoplasm, the reduction of Cr(VI) to Cr(III) by antioxidants such as ascorbate, glutathione, and cysteine occurs in a step-wise manner (dependent on the antioxidant) and generates reactive chromium intermediates as well as reactive oxygen species capable of exerting oxidative macromolecular damage. Remnant Cr(VI) is transported to the nucleus, where it is reduced to Cr(III), causing oxidative DNA damage. Cr(III) forms DNA adducts, protein-DNA crosslinks and causes general genomic instability.

Oxidative stress

Oxidative stress is a major contributor of the initiation and progression of carcinogenesis through the overproduction of reactive oxygen species (ROS) which can cause oxidative damage to proteins, lipids, and DNA[11]. While cells have evolved mechanisms to mitigate ROS-mediated damage, reduction of intracellular Cr(VI) can shift the state towards imbalance with evidence suggesting that the reactive intermediates can undergo Fenton-like and Haber-Weiss reactions to further enhance production of free radicals[12], [13](Figure 1). The oxidation of DNA generates several different types of lesions and the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is commonly used as a biomarker for oxidative stress[14], [15]. 8-oxodG sites are primarily repaired through the Base Excision Repair (BER) pathway following the removal of the oxidized guanine by OGG1[16]. Excision of 8-oxoG results in the formation of an abasic site and introduction of a single-strand break, which is then further processed by additional repair enzymes[17]. Recently, Poetsch has written an extensive review describing how inefficient repair of DNA lesions induced by oxidation can lead to alterations in the conformation of DNA and disruption of protein-DNA interactions, mutations, and genomic instability[18]. Accumulation of 8-oxodG may promote mutagenesis through G:C to T:A transversions while the inefficient repair of abasic sites can result in mutations and formation of double strand breaks[19].

In vitro studies have shown that byproducts of Cr(VI) reduction promote the formation of abasic sites and single-strand breaks[20]. Furthermore, the Sugden group suggested that the initial 8-oxoG sites can be further oxidized into highly toxic lesions, spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh), capable of inducing G:C to C:G transversion mutations and preventing elongation of RNA Polymerase II on account of its structural rigidity[21], [22](Figure 2). Exposure to chromates in occupational workers has been associated with increased levels of DNA double-strand breaks, micronucleus formation, and 8-OHdG[23], [24]. Additionally, Xia et al. reported that mRNA levels of the Base Excision Repair (BER) enzyme hOGG1 are reduced in exposed workers, with further in vitro analysis demonstrating a dose-dependent reduction in hOGG1 mRNA that could return to baseline levels following removal of the challenge[25]. Together, these results suggest that Cr(VI) can potentiate genotoxicity through the increased production of ROS and suppression of critical enzymes involved in repair of oxidized DNA lesions.

Figure 2:

Hexavalent chromium induces diverse chromatin alterations. a) Once Cr(VI) reaches the nucleus, it causes damage through direct oxidation and formation of Cr-DNA adducts. This evokes a DNA damage response that brings alongside it, chromatin structural changes to allow for DNA repair. Additionally, chromium mediates protein-DNA crosslinks that impair protein function. b) Once polymerases encounter these bulky DNA lesions, they largely pause, inducing a state of transcriptional disruption (RNApolII pausing). c) Cr(VI) alters chromatin accessibility in flanking regions of transcription factors CTCF, AP-1 and BACH1. This can be attributed to altered transcription factor binding dynamics, through as-yet unknown mechanisms (d); or by direct nucleosomal displacement (e). Chromium alters nucleosome arrangement, resulting in position shifts and occupancy changes.

Chromium adducts and genomic instability

While Cr(VI) does not directly interact with DNA under physiological conditions, its intracellular reduction results in the accumulation of Cr(III) and subsequent formation binary (Cr-DNA) and ternary (ligand-Cr-DNA) adducts across the genome(Figure 1). Multiple groups have sought to better understand the mechanisms of adduct formation which is thought to occur through the coordination of Cr(III) with the phosphate backbone of DNA[3], [26]. Interestingly, there are published studies suggesting that interactions with nucleobases as well as the phosphate group may be implicated in the chelation of chromium[27], [28]. The resulting lesion does not appear to cause significant distortion of the DNA structure itself [29]. Given that chromium can form both binary and ternary adducts, this raises the question whether formation of these lesions is the result of independent mechanisms or a step-wise process in which binary adducts progress to ternary adducts. To this end, studies investigating the mechanistic steps involved in ternary adduct formation have supported the hypothesis that Cr(III) first interacts with DNA to generate a binary adduct which can then progress to a ternary adduct in the presence of an appropriate ligand[30]. Together, these studies suggest that the intracellular reduction of Cr(VI) generates Cr(III)-DNA adducts across the genome primarily through interactions with the DNA phosphate backbone which, if left unrepaired, can crosslink additional ligands and generate ternary adducts.

Current evidence suggests that the protective mechanisms of antioxidant-mediated reduction outside the cell are also critical components in the formation of ternary adducts which are more toxic than their binary counterparts. The majority of adducts in vivo are expected to be the ternary form and consist of ascorbate, glutathione, cysteine, or histidine despite the abundance of binary adducts in vitro[31]. Studies performed by the laboratory of Anatoly Zhitkovich have demonstrated that the restoration of ascorbate, the principal reduction agent, in cultured cells results in an altered response to Cr(VI) treatment and increased cytotoxicity, suggesting that ascorbate-driven reduction is a major contributor to Cr(VI)’s carcinogenic potential[3], [32], [33].

Adducts can be efficiently resolved through the excision repair pathways, but these processes have also been implicated in promoting chromosomal instability[34], [35]. Of particular interest, the system responsible for error correction during DNA replication, mismatch repair (MMR), is implicated in the rapid formation of double-strand breaks (DSBs) following multiple rounds of cellular divisions and is considered to be a critical step in the early stages of Cr-mediated carcinogenesis[36], [37]. MLH1, a tumor suppressor protein involved in MMR, is frequently inactivated in lung cancer samples of chromate workers and is associated with increased rates of microsatellite instability suggesting that the initial increase in DSBs leads to a selection process for cells deficient in MMR which are prone to increased rates of spontaneous mutagenesis and genomic instability[38]–[40].

Continued exposure to Cr(VI) frequently results in ultra-structural changes that have been extensively studied by the Wise lab. They have found that chronic exposure to Cr(VI) is associated with increased rates of aneuploidy and centrosome amplification that occurs in a time and concentration dependent manner[41]–[43]. Chromosomal aberrations have been reported in multiple studies following chromate workers and include increased rates of double-strand breaks, microsatellite instability, and binucleated cells with micronuclei[44], [45]. Consequentially, it has been proposed that inactivation of MMR is a critical step in the progression of Cr-mediated carcinogenesis which may contribute to the accumulation of DNA damage and increased rates of cell division failure, ultimately resulting in improper chromosomal segregation and the enhanced risk of aneuploidy[40][46][47][42][48][49][36][50][33]. It is important to mention that microsatellite instability is overrepresented in euchromatin, especially in intronic regions, (relative to heterochromatic and nucleosome protected DNA) resulting in altered gene transcription of targeted genes. Since the DNA damage spectrum is associated with lower expression of the damaged genes, it is likely that the MMR genes are specifically repressed by DNA damage in their vicinity, or on their coding sequences[51]. These lines of evidence support the idea that Cr(VI) mediated DNA damage is closely related to chromatin architecture.

Chromatin architecture alterations as a result of Cr(VI)-mediated DNA damage

Cells mount a swift DNA damage response to resolve potentially detrimental DNA strand breaks. These repair proteins require access to the damaged DNA loci through a carefully orchestrated process of nucleosome disassembly, histone modifications and general organizational changes to facilitate efficient access of the DNA repair site by DNA repair proteins; reviewed here [52]. Thus, Cr-mediated DNA damage has the potential to alter chromatin organization in a profound way.

Briefly, at the DSB locus, the mre11-rad50-nbs1 (MRN) complex directly binds to the damaged DNA ends[53]. The MRN complex then recruits the ATM kinase that phosphorylates the DNA repair proteins as well as histone H2A.X to γH2A.X[54]. The γH2A.X signal binds MDC1, and not only flanks the damaged site, but is propagated along the chromosome, even in trans, to other chromosomes to extend for hunderds of kilobases[55]. MDC1 then recruits the ubiquitin ligases RNF8 and RNF168 that ubiquitinate chromatin and engage the BRCA1 and 53BP1[56]. Next in line is the NuA4-Tip60, a 16 sub-component protein complex nucleosome remodeling complex that acetylates H4 via Tip60 and swaps H2A.Z onto nucleosomes via p400, a SWI/SNF ATPase[57]. Overall, DSBs promote chromatin access though histone acetylation and nucleosome remodeling. Accordingly, the histone deacetylase inhibitor trichostatin A is known to facilitate DNA repair[58]. It is important to mention that Cr(VI) reduces cellular levels of H4K16 acetylation through downregulation of MOF acetyltransferase[59]. MOF and Tip60 acetyltransferases belong to the MYST family of proteins and MOF-mediated H4K16 acetylation is critical for DNA repair[60]. This suggests that Cr(VI) impedes DNA repair, in a manner similar to MMR protein downregulation(discussed above), a mechanism that may be important in selection of mutant clones. The impact of Cr-induced DNA damage on higher order chromatin architecture has not been studied. However, hypotheses can be drawn from other agents that cause extensive DNA breakage. For example, irradiation elicits preservation of chromatin territories in lymphoblasts and fibroblasts. This general preservation of chromatin compartments happens in the context of weakened/decreased distal interactions, mainly to preserve 3D chromatin architectuire while restorative processes are ongoing[61]. This scenario may or may not hold in chromium-exposed cells, because Cr(VI), at low concentrations induces alterations in essential chromatin factor binding and nucleosomal architecture (discussed below).

During homologous recombination, repair efficiency is based on enhanced DSB mobility. This involves coordinated relocation of the damaged DNA strand within the nuclear volume during homology searching. This process is mediated by Rad51, a protein that forms a protective nucleofilament complex with the single stranded DNA[62]. Since homology search is influenced by chromosomal and nuclear organization[52], Cr(VI)-altered nucleosomal occupancy (discussed in the subsequent section) or spatial rearrangement of chromatin could possibly influence DNA mobility during DNA damage response and lead to a delayed DNA damage repair process. Furthermore, Cr(VI) suppresses Rad51 via downregulation of E2F1, Rad51’s primary transcription factor. In the end, Cr(VI) reduces the number of Rad51 foci[63], and induces cytoplasmic accumulation of RAD51 after prolonged exposure[64]. The downregulation and mis-localization of RAD51 in the cytoplasm inhibits RAD51 from participating in homologous recombination repair and possibly hampers the HR process.

DNA-protein crosslinks (DPC) stall essential, chromatin-based processes including gene transcription and DNA replication[65](Figure 2). As such, Cr(VI)-mediated crosslinking is largely responsible for curtailing the expression and stimulus response of highly inducible genes[66]–[68]. Whether specific groups of proteins are targeted for crosslinking is an open question. However, previous studies in our group showed that Cr(VI) crosslinks the repressive DNMT1 and HDAC1 to the promoter of the inducible xenobiotic metabolizing gene Cyp1a1, blocking RNAPII transcription and ligand-mediated induction[69]. It is important to note that during the DPC repair process, cross-linked proteins are degraded to allow for DNA repair[70]. This implies that proteins that are found in these DNA-Cr-protein lesions could lose function, and lends some support to evidence that shows global demethylation[71]–[73], and loss of proteins involved in regulation of chromatin architecture. Additionally, given the complex repair pathway, DPCs are repaired at a much slower rate than DNA breaks, which could allow for sustained effects on chromatin and transcriptional output. Ordinarily, the non-covalent protein-DNA bonds allow for chromatin flexibility and mobility, properties that are required for the dynamicity of chromatin structure and response to the environment in gene regulation. DNA-protein crosslinks however, are likely to impair flexibility of higher-order chromatin structure, including the apposition of enhancer and promoter contacts for transcriptional activation and long-distance chromatin interactions.

Direct effect of Cr(VI) on chromatin and functional outcomes of disrupted chromatin architecture

Gene transcription is controlled through several layers of regulatory mechanisms that include DNA accessibility and methylation, post-translational histone modifications, and three-dimensional chromatin interactions. Hexavalent chromium exposure is associated with altered gene expression profiles and its disruption of the epigenome is an important contributor to the progression of carcinogenesis.

Recent advances in our understanding of the genome’s spatial organization highlight its importance in the maintenance of the transcriptome. At the highest level, chromosomes are arranged in a non-random orientation and occupy chromosomal territories, which are then further segmented by additional chromatin-chromatin interactions into topologically associated domains (TADs)[74], [75]. These functional domains provide spatial control for the regulation of gene expression through insulation of histone modifications and orchestration of enhancer-promoter interactions. Rewiring chromatin interactions is implicated in multiple diseases and cancer progression and further study in the context of chromium exposure will help to bridge the gap between increased structural abnormalities and functional outcomes[76].

Despite the global distribution of Cr-DNA adducts across the genome, double-strand breaks are targeted to regions of euchromatin, highlighting a potential role for suppressing genes that active in response to exposure[9](Figure 2). When considered together, the evidence shows that high Cr(VI) concentrations induce a spectrum of DNA lesions. While on the other hand, short-term exposure to lower doses could have other effects on chromatin dynamics.

Within our own group, we have sought to better understand how chromium exposure influences transcriptional regulation through multiple mechanisms. In accordance with earlier findings, chronic Cr(VI) exposure inhibited the expression of multiple tumor suppressor genes[77]. Additionally, Ovesen et al. reported that long-term passaging of murine hepatoma cells in the presence of low concentrations of Cr(VI) resulted in the accumulation of DNA damage and altered transcriptional response to a common co-toxicant, benzo[a]pyrene[78]. In a targeted study of the extended mouse Cyp1a1 promoter, Schnekenburger and colleagues demonstrated that Cr(VI) treatment crosslinks the repressive complex histone deacetylase 1-DNA methyltransferase 1 (HDAC1-DNMT1) and maintains an inhibitory chromatin state capable of suppressing the induction of expression in the presence of an AHR-activating ligand[67], [79]. Taken together, these findings suggest that the regulatory mechanisms of chromatin accessibility may be primary targets of chromium toxicity.

To better assess how disruptions in chromatin accessibility are associated with changes in transcription, our group performed FAIRE-seq in parallel with RNA-seq to profile the association between transcription and differentially accessible regions following both low and acute Cr(VI) treatments. Interestingly, low concentrations exhibited distinct profiles of both chromatin state and gene expression profiles. Of note, AP-1, BACH2, and CTCF were transcription factors enriched in differentially accessible regions and these results were subsequently confirmed using additional complementary sequencing techniques[10], [80](Figure 2). We particularly observed Cr(VI)-induced alterations in nucleosomal arrangement, that were characterized by nuclesome occupancy changes, position shifts of 10 bases or more, and changes in position amplitude(Figure 2). In agreement with our previous study using FAIRE, this disruption occurred at chromatin regions enriched for CTCF and AP-1 binding sites[10]. These changes indicated an ‘opening up’ of transcription factor binding sites, indicating the absence of a protein or vacation of actual nucleosomes and increased insertion frequency in motif flanking sequences. These finding support the notion that Cr(VI), even in low doses, has significant impact on chromatin structure through disruption of basic nucleosomal arrangement.

CTCF, in conjunction with the cohesin complex, is an instrumental component involved in the three dimensional organization of chromatin and facilitates the formation of chromatin loops and TADs[81], [82]. Systematic analysis of CTCF ChIP-seq profiles in human cancers by Fang et al. [83] found that the context of cancer-specific binding events are dependent on the region of the genome in which they occur and are associated with changes in chromatin interactions and can be modulated by the activity of other transcription factors. In agreement with their conclusions, our characterization of differentially-bound CTCF in Cr(VI)-treated cells using ChIP-seq found that the gain or loss of CTCF was dependent on regional context. In particular, sites exhibiting reduced CTCF binding were enriched in the promoters of moderately active genes associated with functionally relevant biological processes, suggesting that areas of active gene transcription may be prone to Cr-mediated disruption[84]. In order, to characterize Cr(VI)-mediated effects on higher-order chromatin organization, we utilized computational algorithms that predict chromatin contacts based on ChIP-seq data. Although we did not identify alterations in number or length of loops, we found that CTCF chromatin loop anchors that showed reduced CTCF binding upon Cr(VI) exposure had higher initial levels of CTCF ChIP-seq signal, while anchors that enriched CTCF upon Cr(VI) had a lower level of CTCF binding[84]. Thus it appears that Cr targets regions with a strong CTCF presence and supports the notion that sites with significantly depleted CTCF are likely to be strong loop anchors or are important for the maintenance of chromatin interactions. Importantly, the CTCF loss at these sites was matched with an increase in H3K27acetylation, meaning that these sites could serve as enhancers or in mediating enhancer-promoter contacts. While it is evident that Cr(VI) alters the binding profile of CTCF and the accessibility around CTCF, AP1 and BACH1 motifs, it remains unclear how or if changes in binding affinity of these proteins arise from direct interaction of chromium species with their heterodimerization or DNA binding motifs. CTCF bears eleven zinc finger DNA binding domains that coordinate docking and DNA loading. A previous study that used a thiolate complex to simulate a 2-cysteine, 2-histidine zinc finger showed that Cr(V) and Cr(VI) displace of Zn(II) from the zinc finger[85]. In addition, Raja and Nair showed that Cr(III) complexes with varying charges impair the DNA binding capacity of Sp1 and TFIID, effectively reducing transcription of a reporter gene [86]. It is noteworthy that Sp1 contains a zinc finger DNA binding motif implying that generally, chromium could interfere with the function of zinc finger proteins. On the other hand, the AP1 protein complex features the leucine zipper (bZIP) heterodimerization motifs that bifurcate into a DNA binding terminal. The positively charged amino acids lysine and arginine are responsible for DNA binding. Whereas there is no direct evidence of Cr(VI) binding to any of the bZIP transcription factors, we posit that there could be a possible direct interaction that alters protein function, either in DNA binding or dimerization. This idea arises from the fact that the trivalent, pentavalent and hexavalent Cr species readily interact with leucine and lysine amino acid residues [87], [88] as well as histone protein that are known to be rich in lysine[89]. AP1 is important for enhancer selection and formation of dynamic chromatin during macrophage differentiation[90]. Cr(VI) could therefore disrupt chromatin organization by altering the DNA binding capacity of proteins involved in regulation of chromatin structures(Figure 2).

Although there possibly exists a degree of stochasticity in Cr-mediated effects, most alterations are predominantly found in euchromatin, specifically in transcriptionally active regulatory and functional regions, resulting in significant transcriptional impact.

Discussion and future direction

The carcinogenic mechanisms of inhaled hexavalent chromium are well documented in occupational settings with additional studies supporting evidence for carcinogenic potential through other routes of exposure, however the mechanisms promoting carcinogenesis are complex and require further elucidation. Genotoxicity appears to be an innate property of Cr(VI) and recent advances by several groups highlight the duality of a cell’s protective mechanisms in the presence of Cr(VI); the same detoxification pathways that are protective in the extracellular environment also potentiate the accumulation of Cr(III) and its toxicity inside the cell. The binary Cr-DNA adducts that form as a result do not create significant distortions of the helical structure itself, but rather serve as a staging ground for the formation of ternary adducts in the presence of reducing ligands. The same DNA repair pathways responsible for resolving Cr-DNA lesions are also implicated in the promotion of genomic instability through the formation of DNA double-strand breaks, which serve as a hallmark of exposure. Concurrently, Cr(VI) exposure has been shown to inhibit the expression of multiple tumor suppressors and alter the epigenomic profiles of several histone modifications, suggesting that chromium must be implicated in the disruption of functional machinery involved in the regulation of transcription. Aberrations in chromatin structure and functional consequences are intertwined and, as a result, highlights the importance of better understanding how chromium’s manipulation of one affects the integrity of the other.

The importance of the continued studies elucidating molecular mechanisms involved in Cr-mediated carcinogenesis can be viewed through two different lenses. From a practical perspective, having a better understanding of both structural and functional disruptions in a unified context may help in the identification of potential biomarkers of exposure as well as add to the body of evidence used in the determination of public health and worker safety recommendations. From a conceptual point of view, integrated analysis chromium’s disruption of chromatin architecture as it pertains to function is an important step in enhancing our understanding of how environmental toxicants target several levels of genomic regulation in order to achieve a particular outcome. Chromatin-chromatin interactions and the organization of the three-dimensional genome are critical aspects of proper transcriptomic regulation and the study of structural perturbations in the presence of hexavalent chromium may provide contextual insights that are instrumental in bridging the gap between mechanisms of genomic instability and the dysregulation of the transcription.