Sex differences in chromatin structure within the dorsal root ganglion

The list of variables responsible for pain sensitivity in a given individual are far too numerous to describe here but encompass the complex interactions of developmental, psychological, psychosocial, experiential, brain regional interactions, and gene expression programs in neuronal and non-neuronal cells. Here, we discuss the paper by Franco-Enzástiga et al.,3 entitled “Epigenomic landscape of the human dorsal root ganglion: sex differences and transcriptional regulation of nociceptive genes,” which explores chromatin accessibility in dorsal root ganglia (DRG) as a potential contributor to differences in transcriptional gene expression and pain sensitivity between males and females. DNA within cells is not a naked double helix, but rather is bound to nucleosomes, composed of histone proteins, which is partly responsible for condensing the ∼6 feet of linear human DNA into each of our cells.2 Importantly, chromatin–DNA interactions comprising the nucleosome architecture play a role in either inhibiting or allowing gene expression by controlling the accessibility to the transcriptional machinery.6 At a mechanistic level, nucleosomes can inhibit gene expression by literally blocking access of transcription factors and RNA polymerase to gene promotor and/or enhancer sequences as well as the structural genes themselves. In contrast in an open nucleosome-free configuration, the assembly of the transcriptional apparatus occurs unimpeded allowing gene expression to proceed (see Fig. 1 and author's Fig. 1B).Figure 1.: (A) Illustration of ATAC-seq and nucleosome arrangement for closed and accessible DNA. The DNA helix is wound around nucleosomes (blue ovals), which are composed of 4 histone proteins (see panel B). The open chromatin can have transcription factors and RNA polymerase II bound at or upstream from the TSS. The occupancy of the open region DNA by transcription factors can also block cutting by Tn5 nuclease (not shown). Thus, the open region at the TSS provides 2 types of information: first the identity of the gene downstream from the TSS, and second presence of transcriptional regulatory factors and their DNA binding motifs in the upstream promoter region, which provides information on how gene expression is controlled. (see Fig. 3 in Franco-Enzástiga et al.3). (B) CryoEM analysis of transcription of a synthetic DNA template. DNA is being unspooled from the nucleosome and fed into the transcription slot of a eukaryotic RNA polymerase II (green strand) and in then rewound subsequently (yellow strand). This shows the transcriptional apparatus in a simplified experimental situation and allows visualization of the molecular structural interactions not possible with a diagram.1 ATSA, Assay for Transposase-Accessible Chromatin using sequencing; TSS, transcription start sites.Although previous studies have examined chromatin accessibility using low-throughput techniques on a gene-by-gene basis such as by DNAse footprinting, the study by Franco-Enzástiga et al., used a high-throughput genome-wide technology called Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Assay for Transposase-Accessible Chromatin-seq uses an enzyme, the Tn5 transposase that fragments DNA only in nucleosome-free regions, at the same time the enzyme attaches barcoded primers to both ends of the Tn5-generated DNA fragments. The tagged fragments are amplified by polymerase chain reaction, sequenced en masse, and mapped back to the corresponding sequences in the genome to define active gene regions. Here, Franco-Enzástiga et al., applied ATAC-seq to explore sex differences in genes expressed in the DRG between male and female organ donors. The authors found that approximately 15% of ATAC-accessible DNA regions mapped to promoter sequences around transcription start sites of many different genes. Further analysis of differences in transcription start sites found 3005 differentially accessible chromatin regions (DARs). While in female donors, a majority of DARs were located on the X chromosome, in male donors, the DARs were associated with autosomal chromosomes. Genes identified in female donors include MAP7D2, FRMPD4, PORCN, PNMA3, and TKTL1 that encompass a mix of protein functions ranging from microtubule assembly, regulation of synaptic spine density, an O-palmitoyltransferase also involved in plasticity, a paraneoplastic antigen, and transketolase-like protein 1.4 Genes identified in males included a nuclear receptor transcriptional repressor, an interferon regulatory transcription factor, cyclin B1, a serine/threonine kinase, and several transcription factors such as FOXO1. An additional layer of analysis involved identification of transcription factor binding motifs within the nucleosome-free regions of male and female DRG samples. Interestingly in female samples, DNA elements for a number of zinc finger transcription factors (eg, EGR1, EGR3, SP1, SP2, SP4, and SP9) were more abundant compared to male samples. By comparison, in male samples, a greater number of motifs for basic leucine zipper family of transcription factors (eg, JUN, FOS, FOSL2, and ATF4) were detected. These data imply a differential strategy of transcription usage in males and females. This analysis also showed potential regulatory networks of genes coding for cytokines, tumor necrosis factor signaling, interferon regulatory factors, the mechanosensor PIEZO2, and calcium channels. However, the number of male and female samples studied were small, and these results need to be replicated in larger studies before detailed conclusions are made. Despite obtaining a copious amount of chromatin accessibility gene data in the first part of the study, the information was not linked to specific cell types within the DRG. To gain further insight into cell-type chromatin accessibility differences, the authors next used a technique called spatial ATAC. In spatial ATAC, a DRG tissue section was placed on a grid of “tiles,” each containing unique DNA barcodes complementary to the adapters added by Tn5 and captured onto the tiles apposed to a cell type in the DRG section. This is an elegant technique; however, the amount of sequence information obtained from any one cell is relatively sparse. To overcome this limitation, cells with similar gene profiles can be clustered. Eight broadly defined cell clusters were assembled. Cluster 1 was classified as neuronal based on abundance of chromatin accessibility in SNAP25, a gene encoding a synaptic vesicle protein. As might be expected, this cluster also shows chromatin accessibility for the gene coding for calcitonin gene–related peptide precursor (CALCA) and the NaV1.8 sodium channel (SCN10A). Other clusters identified were non-neuronal cells ranging from satellite glia to myelin-producing Schwann cells. The authors identified a number of sex differences in accessibility for some neuronal genes (eg, (transient receptor potential V3) TRPV3; implicated in thermal heat transduction)7 in DRG neurons, but additional studies are needed to ascertain whether these transcripts are differentially expressed between males and females. It is worth noting that not all genes detected with this method are well expressed in DRG neurons as measured by RNA-Seq. This discordance suggests that while some genes show measurable chromatin accessibility, they are not all transcribed. One possibility is that despite low expression for a given gene, an accessible chromatin state could be indicative of readiness for on-demand gene induction in response to a physiological or pathophysiological state change. From this study by Franco-Enzástiga et al., and many others, it is clear that sex differences in pain sensitivity remain complex, but the beginning of a genetic map to address this question is emerging. Clearly ATAC and spatial ATAC provide yet another set of tools to probe gene regulatory mechanisms in sensory neurons. Ideally, a more relevant study design would examine whether chromatin accessibility plays a role in pathological pain states. While not readily practical with human DRG samples,5 a variety of animal models could alternatively be used to explore this question with ATAC. Eventually, it may be possible to obtain evidence for a dynamic epigenetic component to chronic pain, providing a unique viewpoint into gene regulation in critical pathogenic processes. Conflict of interest statement The authors have no conflicts of interest to declare.