FOXO3 longevity interactome on chromosome 6

Summary FOXO3 has been implicated in longevity in multiple populations. By DNA sequencing in long‐lived individuals, we identified all single nucleotide polymorphisms (SNPs) in FOXO3 and showed 41 were associated with longevity. Thirteen of these had predicted alterations in transcription factor binding sites. Those SNPs appeared to be in physical contact, via RNA polymerase II binding chromatin looping, with sites in the FOXO3 promoter, and likely function together as a cis‐regulatory unit. The SNPs exhibited a high degree of LD in the Asian population, in which they define a specific longevity haplotype that is relatively common. The haplotype was less frequent in whites and virtually nonexistent in Africans. We identified distant contact points between FOXO3 and 46 neighboring genes, through long‐range physical contacts via CCCTC‐binding factor zinc finger protein (CTCF) binding sites, over a 7.3 Mb distance on chromosome 6q21. When activated by cellular stress, we visualized movement of FOXO3 toward neighboring genes. FOXO3 resides at the center of this early‐replicating and highly conserved syntenic region of chromosome 6. Thus, in addition to its role as a transcription factor regulating gene expression genomewide, FOXO3 may function at the genomic level to help regulate neighboring genes by virtue of its central location in chromatin conformation via topologically associated domains. We believe that the FOXO3 ‘interactome’ on chromosome 6 is a chromatin domain that defines an aging hub. A more thorough understanding of the functions of these neighboring genes may help elucidate the mechanisms through which FOXO3 variants promote longevity and healthy aging.

Suppl. Fig. S2. Interactions between potentially functional FOXO3 SNPS and nearby genes on chromosome 6q21.

(a) Putative functional SNPs in FOXO3, cis-regulatory elements, RNAPII Chip-Seq data), and chromatin contact points for RNAPII.
In part (a) of Fig. S2 the 13 putative functional SNPs of FOXO3 were mapped relative to known transcription factor binding sites (TFBSs) and RNA polymerase II (RNAPII) binding sites (blue) using the WashU Genome Browser (Zhou et al. 2011). The TFBSs are from the Transfac database (green), noting the positions of the 13 SNPs (small vertical black arrows), RNAPII chromatin immunoprecipitation sequencing (ChIP-Seq) data (blue vertical lines), and chromatin contact points for RNAPII using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) (pink loops). This figure shows that there is a high density of enhancer elements in the FOXO3 promoter and that many of these are physically linked to elements located in introns 2 and 3 by RNAPII binding sites. An overlay of FOXO3 is shown (blue arrows). Data were derived from the WashU Genome Browser (Zhou et al. 2011). The RNAPII binding site data were from channels: "Berstein HUVEC Pol2(b)" and "Berstein NHEK Pol2(b)". The RNAPII looping data are from channel "ENCODE GIS ChIA-PET K562 POL@ rep2".
In part (b) of Fig. S2 short-range CTCF chromatin contacts (shown as loops) between the proximal promoter region of FOXO3 (blue bar) and neighboring genes. The promoter region of FOXO3 was found to connect through chromatin contacts with several neighboring genes, including OSTM1, SNX3, SESN1 and CEP57L1. Shown are CTCF binding sites (blue boxes) obtained by ChiP-Seq connected through CTCF-bound chromatin looping (pink loops) obtained by ChiA-PET. Data for CTCF ChIP-Seq are from channels "Bernstein NH-A CTCF" and Berstein GM12878 CTCF". Data from ChiA-PET are from the channel "ENCODE GIS ChiA-PET K562 CTCF In part (c) of Fig. S2 long-range CTCF chromatin contacts (loops) are shown between promoter of FOXO3 (blue bar) and neighboring genes. FOXO3 is at the center of a 7,268,123 bp long segment on chromosome 6 that is connected through CTCF contacts. The same data files as in "(b)" are shown, except that the view has been zoomed out. In part (a) of Suppl. Fig. S3 the region from 107,000,000 to 113,000,000 on chromosome 6 is early-replicating (green peaks). FOXO3 is denoted by the blue arrow. In part (b) the origins of DNA replication are shown by blue bars and appear to peak around FOXO3 (blue arrow). In part (c) there appear to be origins of DNA replication in FOXO3 introns 2 and 3. The blue rectangles denote exons 1-4, while the blue line denotes introns.

Suppl
This Figure shows results of use of the public database "Genomicus" to search for conservation of the region surrounding FOXO3. These data are derived from Ensembl release 76, which contains genome annotations for 68 extant species. The primate order is shown in the last 13 rows, predicting a syntenic region that is conserved in the Euteleostomi clade (row 2), which dates back 420 million years. Those conserved genes shown in row 2 include the region spanning the genes AIM1 through METTL24, that contain several gaps, and the region from C6orf203 through PPIL6 (black arrows) that has no gaps. FOXO3 is shown by the green arrow and the human chromosome is the bottom green row labeled "human -Chr:6". Suppl. Fig. S5. FISH experiments to determine the effect of H 2 O 2 stress-induced activation of FOXO3 in lymphoblast cell lines on interaction with two genes (HACE1 and LAMA4) flanking FOXO3 on chromosome 6q21.   S5 shows results of FISH experiments using lymphoblast cell lines derived from 20 subjects who were the offspring of the long-lived Japanese American subjects used for the genetic association studies of FOXO3 SNPs. We measured the distances between fluorescent signals for HACE1 and FOXO3 (GA), between FOXO3 and LAMA4 (AR), and between HACE1 and LAMA4 (GR). Early evaluation and quality control of the FISH probes suggested that relative gene position might affect expression. We therefore chose to also measure the angles between the three probes using FOXO3 as the vertex (GAR).

(a) Explanation of interphase chromosome measurements made. (b) Interphase chromatin organization (i) Distances between genes comparing genotype vs. treatment
(a) shows what measurements were made. (b) (i) shows the mean distances, comparing paired untreated vs. treated cell lines from 10 subjects homozygous for the common allele of FOXO3 (TT), and paired untreated vs. treated cell lines from 10 subjects heterozygous for the longevity-associated G allele (GT genotype). Ratios of distances were also compared in an effort to reduce cell-to-cell variability in nuclei diameter.
(b) (ii) shows total distance (i.e. circumference; GARG, mean±SE). Data were analyzed by ttest. While the differences between TT vs. GT genotypes were not significant (P=0.053), differences between treated and untreated were highly significant (P<0.0001).
(b) (iii) shows confirmation of this relationship by measuring the angles of the three genes to each other for 20 cell lines from different subjects with TT genotype (10 untreated and 10 treated) and 20 with GT genotype (10 untreated and 10 treated).
The results indicated that in the treated cells the three genes were more co-linear (in line with each other).
(b) (v) is a diagrammatic summary of the changes in gene positions that were seen for each genotype for untreated (resting) and treated (stressed) cells. It appeared that, upon activation, FOXO3 became interposed between the two outlying genes, HACE1 and LAMA4, and that the longevity genotype was already closer to that final position than the common genotype. To summarize part (b): • The total distance "GARG" was significantly shortened following treatment.
• The G-A-R angle was significantly increased following treatment.
• Less change was noted in cells carrying the G allele since FOXO3 was more likely to already be in the "induced" orientation. (c) shows the increase in FOXO3 and HACE1 expression in response to treatment of lymphoblast cell lines with H 2 O 2 (part (i), P<0.0001 comparing expression and genotypes by ttest). There was no significant difference in expression of HACE1 with respect to FOXO3 genotype (part ii).