In this study, we mapped genome-wide labeling of chromatin by the SUMO-1 protein throughout the human cell cycle and made multiple discoveries. (i) On a chromosome-wide scale, the SUMO-1-binding profile was consistent during interphase, but changes were evident during mitosis with a decrease in SUMO-1 binding events. (ii) We found the ChAP-seq data of SUMO-1 replicates were highly reproducible, and the pattern of SUMO-1 binding to chromatin was dynamic during cell cycle progression. (iii) The SUMO-1 distribution on the chromatin was enriched on active genes, especially the regulatory elements such as CpG islands and promoters. (iv) SUMO-1 localization on promoter chromatin was highly correlated with the transcriptional activation signal of H3K4me3 and had low correlation with the transcriptional repressive signal H3K27me3. (v) The effect of SUMO-1 labeling of promoters on gene expression was in many cases stimulatory. (vi) Genes that encode ribosomal protein subunits and translation factors were the most significant subgroup stimulated by SUMO-1.
An initial clue that SUMO-1 was correlated with gene activation was that it was associated with highly active promoters throughout interphase, decreased during mitosis when transcription is generally repressed and then was present again in the G1 phase of the cell cycle. It must be recognized with this cell cycle correlation of SUMO-1 marks that the absence of a chromatin mark during mitosis can have many causes aside from the regulation of transcription. It has been shown that SUMO-1 is removed from chromatin during mitosis (42
). Our results are consistent with that earlier finding, though we do still observe SUMO-1 marks on specific sites, for example many promoters (A) including the SLC1A3
gene (A). The results indicate that the signal by SUMO-1 on a promoter is complicated: in many cases it is stimulatory and in others the SUMO-1 tag is repressive (). The genome-wide analysis presented in this study is a first step toward deciphering how SUMO-1 is regulating gene expression. The striking finding on which we focused was that among very highly expressed genes, SUMO-1 is a stimulatory mark (Supplementary Table S4
). From the PCA (D), it is clear that how SUMO-1 associates with a variety of genetic elements changes through the cell cycle, and future analyses are targeted at deciphering these aspects of the complex chromatin signaling by SUMO-1.
Interestingly, when comparing the labeling of chromatin by SUMO-1 in this study with the labeling of chromatin by ubiquitin during mitosis, we found a high level of concordance. The promoters of many genes whose expression is important in the G1 phase of the cell cycle are bookmarked by ubiquitination during mitosis and then de-ubiquitinated in G1 (Arora et al.
, submitted). Of the 3446 promoters found to be bookmarked by ubiquitin during mitosis, 1829 promoters (53%) were labeled by SUMO-1 during interphase (Supplementary Table S5
). These results are most consistent with SUMO-1 having a stimulatory role in regulating gene expression via the chromatin.
SUMOylation of transcription complexes and/or chromatin-modifying complexes is known to regulate subcellular localization, protein–DNA-binding affinity and repress gene transcription. For example, SUMOylation of a variety of transcription factors/ co-factors fused with reporter gene inhibits gene expression (16
). Moreover, expression of a dominant-negative E2 Ubc9, which inhibits SUMO conjugation to substrate proteins, or mutation of the SUMO-targeting sites on transcription factors resulted in upregulated transcriptional activity of specific genes (15
). SUMO-1/2/3 have all been shown to recruit histone deacetylases (HDACs) (50
) and thus repress acetylated chromatin. For these reasons, we were surprised that our global SUMO-1 binding data showed SUMO-1 actually marked constitutively expressed genes. From the genome-wide data, SUMO-1 associates with highly expressed genes that encode proteins involved in protein biogenesis. Whether the SUMO-1 moiety was recruited by specific bound factors or DNA elements is unclear at this time. It is possible that the transcription activation process itself recruits the SUMOylation to highly active promoters. On these high-activity promoters, binding by SUMO-1 is stimulatory.
One published study focused on SUMO marking of multiple promoters in yeast. That study suggested that SUMOylation of the promoter bound factors is associated with constitutive transcription and also activation of inducible genes, and inactivation of SUMOylation in yeast harboring a defective ubc9 gene reduced SUMO at the constitutive promoters and decreased gene expression in yeast (32
). In contrast, in our study, the outcome of Ubc9 depletion is not necessarily consistent with SUMO-1 depletion, and we suggest that this inconsistency is due to SUMO isoforms (i.e. SUMO-2/3) that might have opposing transcriptional activities. The conjugation of SUMO-1 and SUMO-2/3 on substrates has been shown to have an opposing role with a specific transcription factor (53
). In another study, SUMO-1 was located on both active and repressive photoreceptor-specific genes to regulate rod cell development in a mouse model (54
). The results of our study substantially add to the concept that SUMO-1 is a stimulatory mark on chromatin since we found that genome-wide in the human cell, the preponderance of SUMO-1 chromatin marks on, or near promoter regions are associated with active gene expression.
Ribosome biogenesis proteins, such as small nuclear ribonucleoproteins, and ribosomal proteins were identified as novel SUMO targets and were required for nucleolus formation (17
). Moreover, a recent study showed SUMO system is critical for nucleolar partitioning by regulating a novel ribosome biogenesis complex (55
). The current study finds that not only are the ribosomal proteins SUMOylated but also the genes encoding ribosomal proteins and translation factors are labeled by SUMO-1 on the chromatin over their promoters. Taken together, we suggest that SUMO-1 regulates nucleolar integrity during the cell cycle processing, both transcriptionally and post-translationally.
Since impairing SUMO-1 on these promoters resulted in lower expression, this shows that efficient SUMOylation is critical for optimal gene expression. SUMO-1 marking on these translational machinery genes may function to maintain gene expression and protein stability perhaps by antagonizing other repressive chromatin marks or regulating the subcellular localization of partner proteins required for repression. In addition, while SUMOylation plays a critical role on gene repression on a subset of genes, SUMO-1 also has other properties, for example, regulating the assembly of transcription machinery (56
); therefore, SUMO-1 marking on those housekeeping genes may be an early modification affecting chromatin remodeling. It is unclear at this time what are the relevant chromatin proteins in promoters conjugated to SUMO-1. The position of the peak of the SUMO-1 mark on constitutive active promoters is at −200 relative to the TSS. Such a position could be consistent with the −1 nucleosome or close to where the components of general transcription machinery would be expected to bind. A previous study has shown that SUMO-1 post-translationally modifies hsTAF5 in TFIID to modulate TFIID promoter-binding activity (18
). It is possible that this is the factor SUMOylated at promoters in our studies; however, it would be a complicated mechanism by which SUMOylation of a general transcription factor would be associated with the transcription activation process. Further arguing against TFIID components causing the promoter peak of SUMO-1 binding, the methods used in this study had sufficient resolution to map the bound domains and TFIID subunits would be expected to be closer to the TSSs.
In summary, in this study we demonstrated how SUMO-1 marks promoters in the human genome and how it changes through the cell cycle. We found that SUMO-1 labeling of chromatin is dynamic through the cell cycle, and it is associated at promoters with the most actively transcribed genes. While SUMO-1 was not generally associated with all active genes, a very high percentage of the most active genes (49%) had their promoters modified with bound SUMO-1, and it was shown that in many of the housekeeping genes, the SUMO-1 mark on the promoter was stimulatory to gene expression and is critical for the high expression genes encoding translation factors.