Bioinformatic Analysis of Transcriptional Regulation by Nur77 in Central Nervous System and Immune System

Objective The objectives of this work were to nd genes regulated by Nur77 in neurons and to evaluate the possible common role of this transcription factor in neurons and lymphatic cells using published experimentally generated databases of ChIP-Seq and a microarray. We also characterized Nur77 binding throughout the genome. Results We identied 113 Nur77 target genes in neuronal stem cells and 116 in neuronal cells. Cell adhesion and anchoring processes emerged as regulated by Nur77 in neurons and lymphatic cells. We found 9 common genes regulated by Nur77. Finally, we described a signicant distribution of Nur77 binding sites in strong enhancers and active promoters. This work is a rst step to understand the role of Nur77 and its common targets in neurons and immune cells.


Introduction
Nur77 (also known as NGFI-B, TR3, and NR4A1) (1)(2)(3), is a transcription factor encoded by an immediateearly gene, and an orphan member of the nuclear receptor superfamily (4,5). Nur77 has been largely studied in the immune system for its function inducing apoptosis of auto-reactive immature lymphocytes T and modulating the in ammatory response (6,7). In the Central Nervous System (CNS), Nur77 is widely expressed, particularly in brain nuclei that receive dopaminergic and noradrenergic neurotransmission, which regulates the expression of Nur77 (8). Brain pathologies characterized by an imbalance of catecholamine neurotransmission, such as anxiety, addiction, schizophrenia, and Parkinson disease are associated with changes in the expression of Nur77 (9)(10)(11). Despite the growing amount of information about Nur77 in CNS, the lack of knowledge of Nur77 target genes in neurons hinders the study of its function. Here we analyzed public and experimentally generated databases to provide a list of Nur77directly regulated genes in neurons.
Given that Nur77 is involved in the response to synaptic stimulation and control of metabolism in immune and nervous cells (12)(13)(14)(15), we asked if Nur77 regulates the expression of a set of common genes in the nervous and immune systems. Our analysis revealed a group of genes that binds Nur77 on their promoters and cell adhesion and anchoring functions emerged as regulated by Nur77 in both cell types. We also characterized Nur77 binding sites throughout the genome, nding a signi cant distribution in strong enhancers and active promoters, reinforcing the function of Nur77 as a transcriptional activator.
The work presented here is an approach pretending to guide the experimental focus regarding Nur77 investigation, solving in part the problem of lack of knowledge of Nur77 target genes and presenting new functions that can be attributed to this transcription factor in both the immune and nervous systems.
The microarray data was obtained from the work published by Chen et al. in 2014, (GEO Accession; GSE76805) (12). We used ENSEMBL gene annotation for human (hg19) and mouse (mm10) which were directly extracted as TxDb objects though GenomicFeatures and TxDb. Mmusculus.UCSC.mm10.ensGene R packages. Additional genomic annotations of different chromatin states and functional regions were extracted from UCSC Table browser, including the K562 Genome Segmentation by ChromHMM and Ensembl Regulatory Build.

Nur77 binding site characterization
The overlap between the different ChIP-Seq peaks and genomic features were calculated with the GenomicRanges R package using the function ndOverlaps. We intersected the genomic coordinates with K562 Genome Segmentation by ChromHMM and calculated the overlapping enrichment, by normalizing by total coverage of each chromatin state across the genome.
To obtain Nur77 target genes, we used the annotation of TSS and proximal promoters provided by the Ensembl Regulatory build for human (hg19) and mouse (mm10). The annotated TSS and proximal promoters were assigned to transcripts ID from Ensembl annotation if they were located within 2000 nt upstream and 500 nt downstream from a transcript start coordinate. Then, the function ndOverlaps from GenomicRanges was used to nd Nur77 ChIP-Seq peaks that overlapped with TSS or proximal promoters from the Ensembl Regulatory build. We used the biomaRt R package to nd the common gene symbol associated with each of the Ensembl IDs that were assigned to each TSS and proximal promoter that overlapped with a Nur77 ChIP-Seq peak.

Microarray analyses
We computed the differentially expression results from the microarray data published by Chen et al. in 2014 (12), to lter genes that have an adjusted p-value lower than 0.05 and an absolute value of log2 fold change greater than 1. We plotted the results using ggplot2 and highlighting the names of genes for which a Nur77 peak were detected in their promoters.

Ontology analysis
We use the Gene Ontology Consortium server (www.geneontology.org) (18,19) and processed using default parameters with PANTHER analysis tool (20).

Results And Discussion
Nur77 target genes in neurons We considered as Nur77 target genes, those genes that bind Nur77 in a window of -2kb to + 500 bp from TSS in their promoters, according to our re-analysis of ChIP-Seq data of endogenous Nur77 of mouse neural stem cells (NSC) and NSC differentiated to neurons (NC) (17). To select genes in which the binding of Nur77 in uences transcription, we used a microarray of mRNA from mouse hippocampal pyramidal neurons overexpressing Nur77 (12) and selected genes with a signi cant change of expression after Nur77 overexpression (Fig. 1A). We identi ed 113 Nur77 target genes in NSC (Table S.1) and 16 out these 113 genes changed their expression with a fold-change ≥ 2 after Nur77 overexpression (Fig. 1B). In NC, we identi ed 116 Nur77 target genes (Table S.2) and 17 out of these 116 genes changed their expression with a fold-change ≥ 2 after Nur77 overexpression (Fig. 1C). We found 53 Nur77 target genes common to NSC and NC, suggesting that these genes maintain Nur77 binding to their promoter during neuronal differentiation (Table S.3).
Binding of Nur77 on promoters of genes in the immune system and the central nervous system To nd out whether Nur77 exerts a similar function in the nervous and immune systems and its conservation in human and mouse, we compared the binding peaks from two high quality experimentally generated ChIP-Seq databases: 1.  (Fig. 2B). Many proteins encoded by Nur77 target genes, which are common to the nervous and immune system, are ribonucleoproteins and adhesion molecules. This fact was strengthened by the enrichment of Nur77 target genes in nuclear bodies and areas of adherents and anchoring junctions (Fig. 2C).
In the biological process classi cation, Nur77 target genes were enriched in regulation of Endoplasmic-Reticulum-Associated protein Degradation (ERAD) pathway (fold of enrichment 12.91) and regulation of response to endoplasmic reticulum stress (fold of enrichment 8.01). Three GO terms related to interleukin signaling were enriched: interleukin-12-mediated signaling pathway (fold of enrichment 10.11) cellular response to interleukin-12 (fold of enrichment 9.68) and response to interleukin-12 (fold of enrichment 9.49). Nur77 target genes were also enriched in the regulation of protein autophosphorylation (fold of enrichment 9.49) and cell aging (8.22) GO terms (Fig. 2B).
Interestingly, 9 out of the 271 genes were also found in the set of genes that signi cantly changed their expression after Nur77 overexpression in neurons: AGAP3, BIRC5, DYM, ITGB3, KIF21B, MORN5, RREB1, STRIP2 and WEE1, suggesting that these genes are also regulated in the immune system. Previous evidence supports that Nur77 controls the expression of BIRC5 gene (21), validating our results. Further studies are required to fully validate Nur77 control over these genes, both in the nervous and immune system.
Characterization of Nur77 binding sites throughout the genome.
To further characterize the binding pro le of EGFP-Nur77, we compared the enrichment of ChIP-Seq peaks across 15 chromatin states de ned by Ernest et al. for K562 cell line with the peaks of Nur77 (22). We calculated the overlap enrichment across the EGFP-Nur77 peaks and chromatin states for K562 cell line, obtaining a signi cant enrichment of Nur77 peaks across chromatin states associated with transcription (Fig. 3A). The analysis of the coordinates of EGFP-Nur77 peaks, reported by ENCODE, with respect to the nearest transcription start site (TSS), revealed a high frequency of Nur77 binding between +1000 and -1000 nucleotides from TSS (Fig. 3B).
An enrichment analysis using the human chromatin segmentation model generated by Ernst, (2011) showed signi cant enrichment of Nur77 peaks in chromatin states associated with transcriptional regulatory regions, particularly in strong enhancers and active promoters (Fig. 3C). Two of chromatin states are described as strong enhancers (states 4 and 5), which differentiate in the occurrence of speci c chromatin marks and distance to the TSS. Strong enhancer state number 4 presented a higher occurrence of histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 acetylation (H3K9ac) and was closer to TSS than Strong enhancer state number 5. Nur77 was enriched in both chromatin states described as strong enhancers, exhibiting a log2 enrichment greater than 4 in chromatin state 4 ( Fig. 3C). High enrichment of Nur77 binding was also observed in transcriptional transition states of chromatin. These areas presented similar characteristics to transcriptional elongation areas, but with an increased presence of H4K20me1 and H3K4me1 and more sensitive to DNAse (22), suggesting an intermediate state between the promoter activation and effective elongation. In contrast, we found a negative enrichment for Nu77 binding in transcriptional elongation areas. Nur77 was poorly enriched in states numbers 6 and 7, both described as weak enhancers which differ in the occurrence of H3K4me2 and DNase sensitivity (22). Finally, negative enrichment was observed in heterochromatin regions, indicating the absence of Nur77 in inactive areas of the chromatin (Fig. 3C).
Altogether these data indicate that Nur77 is mostly associated with active sites of chromatin, concordant with the role of Nur77 as a transcriptional activator, also con rmed by the high presence of Nur77 in the TSS. Our data also shows that Nur77 binds transcriptional transition areas, suggesting that Nur77 is present in the promoters of its target genes independently of their state (active or inactive). On the other hand, the data suggests that the presence of Nur77 in enhancers would be limited to the active state.
In conclusion, our data analyses show that Nur77 is bound to the promoter of its target genes independently of their transcriptional state (weak, poised or active). In addition, our data suggests that the presence of Nur77 in enhancers is limited to the active state. We propose that Nur77 is always present in promoters but only binds to enhancers when it is upregulated, modulating thus transcription in response to stimuli.
Our analysis showed a strong participation of Nur77 target genes in anchoring and adhesion functions, which is consistent with the previously described roles of Nur77 in the modulation of neurite growth in neurons (13), and in the immune response (14,15), both processes that require interaction between cells and with the extracellular matrix.
Finally, genes found in this work as common targets of Nur77 in the nervous and immune systems are new and undescribed targets of this transcription factor. The work presented here is an approach pretending to guide the experimental focus regarding Nur77 investigation, solving in part the problem of lack of knowledge about Nur77 target genes and presenting new functions that can be attributed to this transcription factor in both the immune and nervous systems.

Limitations
The work presented here is a re-analysis of previously validated databases. However, differences in protocols or the overexpression of Nur77 could generate biases in the analyses. To be sure that genes described here are really modulated by Nur77, we were very restrictive in the selection process, this could lead to an underrepresentation of all genes regulated by Nur77 in neurons. The datasets analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository, https://www.ncbi.nlm.nih.gov/geo/. Access codes are detailed in the manuscript.
All data generated during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests genes. Adherens and anchoring junctions category includes genes of adherens junction, anchoring junction, focal adhesion, cell-substrate adherent junction, and cell-substrate junction GO terms. Nuclear body category includes genes of nuclear speck GO term. Regulation of response to endoplasmic reticulum stress includes genes of regulation of ERAD pathway GO term. Response to interleukin-12 category includes genes of interleukin-12-mediated signaling pathways, cellular response to interleukin-12 and response to interleukin-12 GO terms (Table S.5).* Figure 3 Nur77 binds to active promoters and strong enhancers in K562 cells. Analysis of publicly available database from the anti-EGFP ChIP-Seq experiment in K562 cell line overexpressing EGFP-Nur77 (16