Page 68 - Haematologica March 2020

C. Zhang et al.
SNP rs31528793 affects Lxn promoter activity and hematopoietic stem cell number
The level of a given mRNA transcript is controlled by trans-acting factors and/or cis-acting modulators. Our data suggest that HMGB2 might act as a trans-acting modula- tor to regulate Lxn transcription. Since we previously reported that several SNP identified by us may contribute to the natural variation of Lxn expression,17 we next asked whether any of these SNP is associated with Lxn expres- sion as a cis-acting regulator. SNP rs31528793 is the only genetic variant in the Lxn promoter region (Figure 1A), we thus asked whether it affects the Lxn promoter activity. We made a G to C mutation in the luciferase reporter con- struct containing the Lxn promoter sequence and per- formed the luciferase reporter assay. The G to C change decreased the promoter activity by more than 2-fold (Figure 6A), suggesting a potential suppressive role of this polymorphism in Lxn transcription (Figure 6A, left two columns). Since HMGB2 binds to this region, we next examined whether G/C variant affects HMGB2 binding. The result showed that HMGB2 further suppresses Lxn promoter activity, and C allele still causes nearly 2-fold decrease of the promoter activity. We next performed the EMSA assay and further confirmed the interaction of HMGB2 with the Lxn promoter containing SNP rs31528793 (Figure 6B). These results indicate that SNP rs31528793 influences Lxn promoter activity, with the G allele conferring a high activity, while the C allele is asso- ciated with a low activity. Therefore, the genetic variants of the Lxn promoter add another layer of regulatory mech- anism of Lxn transcription.
We previously reported that Lxn is differentially expressed in HSC of C57BL/6 (B6) and DBA2 (D2) mice, and its expression level is inversely correlated with HSC number.17 It is known that B6 mice carry G allele where- as D2 mice have C allele. We therefore hypothesized that the G allele is associated with the higher promoter activity, high Lxn expression and low HSC numbers, whereas the C allele has the opposite effect. Next, we examined Lxn expression and HSC numbers in B6, D2 and the other two mouse strains, 129X1/SvJ and A/J that carry G allele at the SNP rs31528793 position (http://www. informatics.jax.org/snp/rs31528793). We found that D2 mouse strain had the lowest expression of Lxn and highest HSC number, whereas all the other three strains showed higher Lxn expression and lower HSC number (Figure 6C and D), suggesting that G/C allelic variant could be indica- tive of Lxn expression level and HSC number variation. It is noted that Lxn expression level varies in strains carrying G allele suggesting that other SNP outside of the Lxn pro- moter region may contribute to such variation.
Discussion
Lxn plays an important role in regulating HSC func- tion.17,38 It was originally identified via the natural variation of HSC numbers between B6 and D2 inbred mouse strains in which B6 mice have fewer HSC than D2 mice at a young age. The expression of Lxn is inversely correlated to the size of HSC population, i.e. its level in B6 is higher than that in D2 cells. Therefore, Lxn is a negative regulator of HSC number and its mode of action is primarily through increasing HSC apoptosis and decreasing HSC regenerative capability and proliferation. However, noth-
ing is known about how Lxn is transcriptionally regulated in HSC and other stem cells, or why it is differentially expressed in different inbred mouse strains.
Here, we identified a Lxn upstream regulatory sequence with a strong promoter activity. More importantly, the SNP (rs31528793) in this region significantly affects its promoter activity, and the G allele carried in B6, 129X1/SvJ and A/J mouse strains confers the promoter a stronger activity than the C allele in D2 strain. Genetic variants have been recently identified to play an important role in transcriptional regulation and thereby resulting in gene expression and phenotype variation.9,13-15,40 We thus proposed that the G/C containing promoter might be involved in Lxn transcriptional regulation. Using DNA pull-down and mass spectrometry, we, for the first time, identified a chromatin binding protein, HMGB2, as a novel transcriptional suppressor of Lxn expression. HMGB2 binding was validated by ChIP-qPCR assay in which the endogenous HMGB2 demonstrated a stronger affinity to the Lxn specific promoter sequence. HMGB2 knockdown increases Lxn expression and decreases HSC numbers in both HSC cell line and BM-derived primary LSK cells. This effect was abrogated when the increased level of Lxn was blocked, indicating that Lxn is one of the downstream targets and functional mediators of HMGB2 in HSC. Altogether, these results suggest that both cis- and trans-factors are involved in the regulation of Lxn tran- scription (Figure 6E). In trans-regulating mode, HMGB2 acts as a suppressor for Lxn transcription. In cis-regulating mode, G allele at SNP rs31528793 is associated with stronger promoter activity, a high level of Lxn expression, and a small size of HSC pool. In contrast, the C allelic vari- ant attenuates these effects and Lxn transcription is less responsive to HMGB2, which leads to a lower Lxn expres- sion and an increased stem cell number. Therefore, our work not only identified HMGB2 as a novel transcription regulator of Lxn, but also provides a potential functional significance of SNP rs31528793 in contributing to natural variations in Lxn expression and HSC number. Despite these findings, how H2MGB2 regulates Lxn transcription requires further investigation. We cannot exclude the pos- sibility that HMGB2 directly regulates Lxn transcription as the transcription factor. But it is also likely that HMGB2 acts as a chromatin adaptor or modifier to recruit other transcription factors for the initiation of the transcription process (Figure 6E). This mode of action was shown in the GFI1b transcription during erythroid differentiation process in which the binding of HMGB2 to GFI1b pro- moter enhances the binding of other factors, such as Oct- 1, GATA-1 and NF-Y, which collectively activates Gfi1b transcription.32 In addition, the relationship of HMGB2 binding site to SNP rs31528793, and how they co-ordi- nately or independently regulate Lxn transcription requires further investigation. However, our current data provide more support for the independent regulatory mechanism because of the following observations. Firstly, the suppression extent of HMGB2 on G-containing pro- moter (ratio of “G+ HMGB2” to “G” is 0.47) is similar to that on C-containing promoter (ratio of “C+ HMGB2” to “C” is 0.48) (Figure 6A). These data suggest that suppres- sion of HMGB2 on Lxn promoter activity is independent of allelic variant. Secondly, results of EMSA also show the similar intensity of shifted bands, suggesting that G/C variant does not affect HMGB2 binding (Figure 6B). Lastly, to further confirm binding of HMGB2 to the Lxn promoter
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haematologica | 2020; 105(3)