The Conserved Non-Coding Sequence 2 (CNS2) Enhances CD69 Transcription through Cooperation between the Transcription Factors Oct1 and RUNX1

The immune regulatory receptor CD69 is expressed upon activation in all types of leukocytes and is strongly regulated at the transcriptional level. We previously described that, in addition to the CD69 promoter, there are four conserved noncoding regions (CNS1-4) upstream of the CD69 promoter. Furthermore, we proposed that CNS2 is the main enhancer of CD69 transcription. In the present study, we mapped the transcription factor (TF) binding sites (TFBS) from ChIP-seq databases within CNS2. Through luciferase reporter assays, we defined a ~60 bp sequence that acts as the minimum enhancer core of mouse CNS2, which includes the Oct1 TFBS. This enhancer core establishes cooperative interactions with the 3′ and 5′ flanking regions, which contain RUNX1 BS. In agreement with the luciferase reporter data, the inhibition of RUNX1 and Oct1 TF expression by siRNA suggests that they synergistically enhance endogenous CD69 gene transcription. In summary, we describe an enhancer core containing RUNX1 and Oct1 BS that is important for the activity of the most potent CD69 gene transcription enhancer.


Introduction
In mammals, 95% of the genome is noncoding, and 40% of the noncoding region plays a role in transcription regulation [1]. In combination with gene promoters, cis-regulatory elements contribute to gene transcriptional regulation as enhancers or repressors. Cis-regulatory elements are often identified as conserved noncoding sequences (CNS) with DNase I hypersensitivity. Cis-regulatory elements contain binding sites for trans-acting transcription factors (TFBSs) and serve as centers of epigenetic changes [2,3]. These elements can be located around or within genes and can affect their transcription from as far as megabases [4]. The three-dimensional conformation of the genome seems to be important to promote contacts between distant elements and their target genes. Thus, these sequences form complex regulatory landscapes within the noncoding genome.
Design of dicer substrate small interfering RNA (DsiRNA). All DsiRNAs described were synthetized by IDT (Integrated DNA Technologies, Coralville, IA, USA). Three DsiRNAs were designed for each gene to be silenced in addition to two additional DsiRNAs and a negative control targeting NC1. The genome reference used for DsiRNA design was mm10. Table 3 provides the sequence and cross-reacting properties of DsiRNAs against POU2F1 (Oct1) and RUNX1 mRNAs.

DsiRNA transfection and CD69 characterization.
Mouse EL-4 T cells (10 5 ) were transfected with a 1 µM pool of three DsiRNAs against POU2F1 (Oct1) or RUNX1 in a 1:1:1 ratio or a negative control DsiRNA against NC1. RNA of transfected cells were extracted, and a qPCR was performed to assess mRNA levels of RUNX1 and POU2F1 (Oct1).
Transfected cells were cultured in a 96-well plate at 37 • C with 5% CO 2 for 16 h and then stimulated or not with 10 ng/mL PMA and 500 ng/mL ionomycin for an additional 6 h. Twenty-two hours after transfection, CD69 surface expression was measured by flow cytometry as follows: cells were washed twice in cold 1X PBS and stained for 30 min at 4 • C with 0.5 µg of an α-mCD69/PE-Cy7 monoclonal antibody (Clone H1.2F3, eBioscience 25-0691-81, San Diego, CA, USA). Samples were acquired with a FACSCanto (Becton Dickinson, Franklin Lakes, NJ USA) flow cytometer, and data were analyzed using FlowJo software. The percentage of inhibition of CD69 expression with respect to its expression in the NC-1 control was represented using GraphPad Prism 7 (version 7, San Diego, CA, USA).
Data representation and statistical analysis. Luciferase assay and flow cytometry data were plotted with GraphPad Prism 7. Significant differences between multiple conditions were tested with a nonparametric one-way ANOVA test performed with GraphPad Prism 7.

Mapping of Transcription Factor Binding Sites within Mouse CNS2 Enhancer
In a previous data-mining study of the regulatory features of the CNSs of the CD69 gene, we proposed that CNS2 is the putative main enhancer based on the enrichment of chromatin accessibility, modifications and bound TF [34]. Given the availability of new Chip-seq data, in the present work, we updated the map of TFBSs in the human locus using Chip-seq data from human hematopoietic cell lines available from the ENCODE consortium ( Figure 2A) and also mapped bound TF to the mouse locus using data from murine primary B and T cells available from the ChIP Atlas and ChIPBase v2.0 databases ( Figure 2B) to compare them. These maps showed that the human CD69 3 untranslated region, intron 1, promoter, CNS1, CNS2 and CNS4 and in the murine 3 untranslated region, promoter, CNS1 and CNS2 have a higher density of TFBSs (TFBSs per 100 bp) than the rest of noncoding sequence within the CD69 locus. Thus, the 3 untranslated region, promoter, CNS1 and CNS2 of both species contain numerous TFBSs, which suggests that they may play a particularly important role in the transcriptional regulation of the CD69 promoter. In agreement with our previous observations, mouse CNS2 also shows a higher total number of TFBSs compared to the other regulatory regions within CD69 locus.
In our previous work [34], we subdivided CNS2 into different subregions based on the presence of TFBSs conserved between 6 mammal species and tested their contribution to the enhancer capacity of CNS2. However, that characterization left undefined regions where bound TFs have subsequently been identified and could play a relevant role. Moreover, considering that some regulatory mechanisms may have diverted during evolution and that, consequently, some important TFBS might not be conserved, in the present work, we subdivided the mouse CNS2 into five regions based on the abovementioned updated ChIP-seq data, taking into account all bound TFs, as well as those bound to sites that are not conserved between six mammal species ( Figure 3A). Regions II and III contain more than 70% of TFs bound to non-conserved sites, while regions I and IV contains TFs bound only to conserved BSs. Region V has intermediate characteristics.   factor binding sites conserved among six mammals species, as previously described [34]; grey boxes

Transcriptional Enhancer Capacity of the CNS2 Region and Its Enhancer Core Activity
Then, we assessed the contribution of the different regions to mouse CNS2 enhancer capacity using a luciferase reporter assay in Jurkat cells stimulated or not with PMA plus ionomycin. In this experiment, we compared the capacity of the complete CNS2 sequence (Prom+I-V) to enhance transcription from the mouse CD69 promoter with those of different CNS2 fragments: I+II+III, II+III and I+II (Prom+I-III, Prom+II-III and Prom+I-II). As shown in Figure 3B regions II+III have an inducible enhancer activity similar to that of regions I+II+III, while regions I+II have lower enhancer potential. Therefore, regions II+III seem to contain the most of the relevant enhancing features for transcription for the CD69 promoter upon PMA/ionomycin activation, while those of regions I, IV and V have a more modest contribution.
While region III contains three overlapping BSs, region II has two separate subregions with TFBSs: One with an Oct1 BS and another with GATA+MyoD TFBSs. We further assessed the contribution of the individual regions I, II and III, as well as the subregions of region II separately or in combination with the region I or III, but keeping, within the tested fragment, the original sequence with the endogenous TFBSs distribution. Figure 4 shows that region II (Prom+II) significantly increases CD69 promoter transcriptional activity upon activation, but neither region I (Prom+I) nor region III (Prom+III) increases CD69 promoter transcriptional activity upon activation ( Figure 4B).  The~60 bp sequence of region II containing the Oct1 TFBS (Prom+Oct1) significantly increases the inducible enhancer activity in a similar manner as region II (Prom+II), while the~65 bp sequence containing GATA+MyoD TFBSs (Prom+GATA+MyoD) did not significantly increase the inducible enhancer activity. These results suggest that the subregion that contains the Oct1 TFBS constitutes the most relevant regulatory feature of region II. Moreover, considering that regions I and III alone do not show enhancing activity, these data indicate that the~60 bp sequence containing the Oct1 TFBS is the shortest sequence identified within regions I+II+III that has transcriptional enhancer activity, likely acting as the enhancer core of CD69 transcription. Furthermore, the combination of this sequence with region I (Prom+I+Oct1,~200 bp) had a transcriptional enhancing capacity as high as half of that of region I-III and similar to that of regions I and II together (Figures 3B and 4B). Although the combination of region III with the~65 bp sequence GATA+MyoD (Prom+GATA+MyoD+III,~190 bp) increased the transcriptional activity twofold compared to the sequences containing the BS of each TF individually, it did not reach transcription levels significantly higher than those of the promoter alone. Since GATA and MyoD BS are between Oct1 and RUNX1, it is not possible to test Oct1+RUNX1 without altering the original TFBSs distribution. Thus, considering that regions II+III have almost as much enhancer activity as regions I-III ( Figure 3B), it is suggested that the Oct1 TFBS also needs to cooperate with region III to enhance transcription. Thus, the Oct1 TFBS seems to act as the enhancer core of CD69 transcription and can synergize with either region I or region III or with both of them to achieve greater enhancement of transcription from the CD69 promoter. Altogether, these data suggest a cooperative modular model between the TFs in region II and those in regions I and III.

RUNX1 and Oct1 Silencing Affects the Transcriptional Regulation of CD69
Given the synergy observed between the Oct1 BS and region III and the fact that the latter contains a BS for RUNX1, an important TF during development and homeostasis of immune cells, we evaluated the relative contribution of Oct1 and RUNX1 to CD69 transcriptional regulation. To do so, we silenced them individually or in combination using siRNA in the mouse T cell line EL-4 and analyzed the effect of silencing on surface CD69 protein in unstimulated cells or cells activated with PMA/Ion. Under both conditions, inhibition of RUNX1 and Oct1 individually resulted in a CD69 expression reduction of~40%, and the joint inhibition of both TFs did not further reduce CD69 expression ( Figure 5). Therefore, RUNX1 and Oct1 contribute to enhancing CD69 expression both at resting and after stimulation. Moreover, the fact that the effect of their individual silencing was the same as that of their combined deletion suggests that they need each other to increase CD69 expression, which supports the cooperative model of enhancing transcription.
PMA/Ion. Under both conditions, inhibition of RUNX1 and Oct1 individually resulted in a CD69 241 expression reduction of ~40%, and the joint inhibition of both TFs did not further reduce CD69 242 expression ( Figure 5). Therefore, RUNX1 and Oct1 contribute to enhancing CD69 expression both at 243 resting and after stimulation. Moreover, the fact that the effect of their individual silencing was the 244 same as that of their combined deletion suggests that they need each other to increase CD69

Discussion
In this work, we deepened the understanding of the enhancer function of mouse conserved noncoding sequence 2 (CNS2) on CD69 promoter transcriptional regulation. Using ChIP-seq data, we mapped the TFBS within CNS2 and used the TFBS distribution to define different regions. Then, we tested the effect of these regions on transcription induced by the CD69 promoter in response to PMA/ionomycin activation in luciferase reporter assays. In this way, we defined a minimal sequence with intrinsic enhancer capacity that contained a conserved Oct1 BS that could further enhance transcription if it was accompanied by either a 3 flanking region containing a conserved SRF BS or a 5 flanking region containing conserved RUNX1, GABPA and Elk-1 TFBSs and could enhance transcription to even higher levels in the presence of both of them. These results suggest cooperation between these TFs. We further tested the effect of Oct1 and RUNX1 silencing on CD69 protein expression. It these experiments, the observed synergy seemed even more striking because the effect of each TF depended on the presence of the other.
In previous studies, we observed that among the different CNSs, CNS2 had the most potent enhancer activity on CD69 transcription. In the present work, the mapping of TFBSs on the CD69 locus showed that several of the TFs that have been shown to bind to CNS2 also bind the promoter region, which suggests their importance in the transcriptional regulation of CD69. Despite the fact that there are fewer data available for the TFs bound to the mouse CD69 locus than to the human CD69 locus, similar to humans, mouse CNS2 is also the region with the highest enrichment of TFBSs. Occupancy by specific TF has been described to be a predictor of enhancer activity of a given region [38]. Thus, these results highlight the importance of CNS2 as a transcriptional enhancer of CD69.
The present results regarding the enhancer capacity of CNS2 regions are in agreement with our previous work, in which the regions with the highest enhancer capacity comprises regions I, II and III, described in the current study [34]. In the present study, the further subdivision of this regions into smaller regions allowed us to identify an Oct1 BS-containing minimum enhancer core that can cooperate with either an adjacent region at the 3 end (region I, containing an SRF TFBS) or 5 end (region III, containing a RUNX1 TFBS) or with both of them to reach greater enhancer activity. Interestingly, although region I did not have enhancer activity per se, it could increase that of Oct1 BS. This could be explained by an interaction of TFBSs in region I with the transcriptional machinery that bounds to the region containing the Oct1 BS. Altogether, these data suggest a cooperative modular model of transcription factors that synergistically enhances transcription. Cooperation between transcription factors may occur based on their physical association, a mechanism frequently described for enhancer sequences that affect the transcriptional behavior of several immune system genes [39][40][41].
Although Oct1 has been widely studied as a regulator of transcription in many cell types [42,43], its precise role is still unclear. Oct1 is known to have both activating [44][45][46] and silencing activity [47][48][49]; moreover, this TF has been described to play a role in CD4 + T cell differentiation [50] and memory formation [51], orchestrating the fate and function of CD4 + effector T cells as a switchable stabilizer of the repressed and inducible state [52], acting at hypersensitivity sites that are distant from promoters of target genes in several cases. RUNX1 has been implicated in the development and homeostasis of different immune subpopulations [53].
As is the case for CNS2, many enhancers are located far from their target genes, but are able to specifically communicate with distal promoters over large distances [54][55][56] through the formation of a chromatin loop [57,58]. Thus, chromosome topology is critical for the interactions between distant enhancers and promoters. However, the relevance of particular interactions for target gene transcription is difficult to assess because they have not always been related to functional outcomes [59,60].
Because of the lack of a genomic context in reporter plasmids, luciferase assay data do not reflect the effects of chromosome topology on transcription. However, the luciferase assay can still be used as a predictor of the regulatory capacity of elements that can be further characterized in a more physiologic context. With that aim, we tested the effects of RUNX1 and Oct1 silencing on the expression of the endogenous CD69 gene. Silencing of these TFs separately in the mouse EL-4 T cell line reduced CD69 expression as much as their joint silencing, suggesting that they can enhance CD69 expression, but only if the other TF is also present. This result suggests a mode of action that is completely synergic. Given the predominant enhancer role of CNS2 in CD69 transcription and, within it, of the Oct1 TFBS, and its observed cooperation with region I, we believe that these results might reflect, to some extent, the role of Oct1 and RUNX1 acting on the BSs described in regions I and II of CNS2. However, we cannot rule out that they might also be influenced by the action of these TFs on other BSs within the CD69 locus. The ChIP-seq databases show two RUNX1 BS found in CD69 promoter sequence. However, since these regions have a much more modest enhancing capacity, we propose that the main enhancer effect of RUNX1 is through the BS in CNS2. Future experiments using ChIP, TF silencing and TFBS mutagenesis might shed more light on the role of the studied TF in CD69 transcription in the genomic context.
In summary, our results strengthen evidence of the role of CNS2 as an essential enhancer of mouse CD69 promoter transcription. Despite the need for further studies on the role of CNS2 in the genomic context, to our knowledge, no detailed studies on the composition and function of regulatory elements within this enhancer have been published. Our work is, therefore, a step forward in understanding how CNS2 regulates CD69 expression.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.